Table of Content

28 August 2022, Volume 26 Issue 24 Previous Issue   

For Selected:

Download Citations EndNote Reference Manager ProCite BibTeX RefWorks

Toggle Thumbnails

Select

Osteoblast differentiation after conditional knockout of 3-phosphoinositide-dependent protein kinase-1 gene from bone marrow mesenchymal stem cells Chen Qiaoling, Bai Yiguang, Liu Kang, Lin Tao, Luo Xuwei 2022, 26 (24):  3785-3789.  doi: 10.12307/2022.554 Abstract ( 642 )   PDF (1179KB) ( 113 )   Save BACKGROUND: The research on 3-phosphoinositide-dependent protein kinase-1 (PDK-1) in and outside China is mainly concentrated in endocrinology and oncology. There are no systematic studies and reports on its influence on osteogenic differentiation in orthopedics. OBJECTIVE: To observe the role of PDK-1 in osteoblast differentiation by transfecting PDK-1flox/flox mouse bone marrow mesenchymal stem cells with adenovirus stably expressing cre enzyme (pHBAd-cre-EGFP).  METHODS: Bone marrow mesenchymal stem cells from homozygous PDK-1flox/flox mice were obtained and cultured in vitro. A control group, an empty virus group (pHBAd-EGFP) and a pHBAd-cre-EGFP group were set. In the control group, osteoblast induction medium was used to induce bone marrow mesenchymal stem cells. In pHBAd-EGFP group, pHBAd-EGFP was used to transfect bone marrow mesenchymal stem cells. In the pHBAd-cre-EGFP group, the recombinant adenovirus containing Cre recombinant enzyme (pHBAd-cre-EGFP) was used to transfect with the PDK-1 gene in bone marrow mesenchymal stem cells followed by osteogenic induction. Alkaline phosphatase staining, alkaline phosphatase activity assay, alizarin red staining, and qPCR were used to detect osteogenesis-related gene expression and to evaluate the differentiation and maturation of osteoblasts.  RESULTS AND CONCLUSION: The alkaline phosphatase secretion, alkaline phosphatase activity, and the ability of cell mineralization in the pHBAd-cre-EGFP group were significantly lower than those in the other two groups (P < 0.05, P < 0.01), and the expression levels of Runx2, osteocalcin, and collagen I were also significantly lower than those in the other two groups (P < 0.01). The results have confirmed that interference with PDK-1 gene in vitro can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells.  Figures and Tables | References | Related Articles | Metrics

Select

Osteogenic differentiation of bone marrow mesenchymal stem cells in obese mice Chen Chichi, Zhang Yu, He Jiachen, Shi Qin 2022, 26 (24):  3846-3851.  doi: 10.12307/2022.564 Abstract ( 614 )   PDF (1917KB) ( 524 )   Save BACKGROUND: The differentiation of bone marrow mesenchymal stem cells is affected by many factors. The bone marrow mesenchymal stem cells of obese mice have better osteogenic differentiation ability.   OBJECTIVE: To explore the difference of osteogenic differentiation of bone marrow mesenchymal stem cells from obese mice induced by high fat diet and normal diet mice. METHODS:  Balb/c mice aged 4-6-weeks were randomly divided into normal diet group and high-fat diet group for 20 weeks. Bone marrow mesenchymal stem cells were isolated by whole bone marrow culture. After 7 days of osteogenic induction, alkaline phosphatase staining and qRT-PCR were performed to detect the expression of osteogenic related genes. At 14 days after osteogenic induction, alizarin red staining was performed. After 20 weeks of high-fat feeding, changes of bone mass of femur were analyzed by Micro-CT. The decalcified sections of the mouse femur were stained with hematoxylin and eosin.   RESULTS AND CONCLUSION: (1) Compared with the mice in the normal diet group, after 20 weeks of high-fat feeding, the weight of the mice increased significantly. (2) Compared with the bone marrow mesenchymal stem cells of mice in the normal diet group, the expression of alkaline phosphatase and the number of calcium nodules in the bone marrow mesenchymal stem cells of mice in the high-fat diet group increased significantly, and the expression levels of osteogenic related genes alkaline phosphatase, osteopontin and RUNT-related transcription factor 2 elevated. (3) The results of Micro-CT reconstruction and bone morphological parameter quantification showed that compared with the normal diet group, the high-fat diet group had a significant increase in bone mass, including bone mineral density, bone volume fraction, bone area to bone volume ratio, bone trabecular thickness, and bone trabecular number increased significantly, while the trabecular separation significantly reduced. Hematoxylin-eosin staining showed that there were more trabecular bones in the femur of the high-fat diet group. (4) It is concluded that osteogenic differentiation ability of bone marrow mesenchymal stem cells in obese mice is enhanced, which further leads to a significant increase in bone mineral density, bone trabecular thickness, and bone trabecular number, and also in the bone mass in mice. Figures and Tables | References | Related Articles | Metrics

Select

Osteogenic differentiation potential of microencapsulated transgenic bone marrow mesenchymal stem cells cocultured with osteoblasts You Wulin, Huang Guicheng, Wang Jianwei 2022, 26 (24):  3852-3857.  doi: 10.12307/2022.565 Abstract ( 534 )   PDF (1832KB) ( 92 )   Save BACKGROUND: With the rapid development of transgenic technology, gene therapy and microencapsulation technology are combined to form microencapsulated gene delivery technology. In addition to providing a good three-dimensional microenvironment for the growth of stem cells and ensuring the large-scale in vitro culture of stem cells, microencapsulation can also use selective permeability membranes to isolate the graft from the host immune system, and effectively avoid immune rejection during allogeneic transplantation. OBJECTIVE: To observe proliferation activity and osteogenetic differentiation potential after coculture of microcapsuled genetically modified bone marrow mesenchymal stem cells (BMSCs) and osteoblasts. METHODS: APA-Foxc2-BMSCs microcapsule complexes were prepared using pulsed high voltage electrostatic microcapsule preparation instrument. Acridine orange/ethidium bromide staining was conducted. Microencapsulated transgenic BMSCs and osteoblasts were cocultured. At 3 weeks after coculture, MTT assay was performed to detect cell proliferation. At 1 week, quantitative detection of alkaline phosphatase activity was performed. At 1 and 2 weeks, qualitative detection of alkaline phosphatase and von Kossa staining were conducted. At 2 weeks, western blot assay and real time PCR were performed to measure osteogenic factor expression. RESULTS AND CONCLUSION: (1) Acridine orange/ethidium bromide staining demonstrated that 70%-80% of the cells in the microcapsule dyed green fluorescence. Under the microscope, cells did not escape from the microcapsule or stain outside the capsule. After breaking capsule, cultured BMSCs grew well again. (2) In the microencapsulated coculture group, alkaline phosphatase staining showed many tan particles in the cytoplasm. Alkaline phosphatase activity was significantly increased, and calcium matrix deposition appeared. (3) In the microencapsulated coculture group, expression of type I collagen, platelet-derived growth factor protein and mRNA significantly increased. (4) The results suggest that microcapsuled transgenic BMSCs cocultured with osteoblasts can strengthen BMSCs proliferation activity and promote BMSCs osteogenic differentiation.  Figures and Tables | References | Related Articles | Metrics

Select

Ouabain induces in vitro differentiation of human bone marrow mesenchymal stem cells into osteoblasts Cui Yinpeng, Guo Ai, Ma Lifeng, Liu Zhenjiang 2022, 26 (24):  3796-3801.  doi: 10.12307/2022.556 Abstract ( 531 )   PDF (2381KB) ( 105 )   Save BACKGROUND: As a ligand of Na+/K+-ATPase, ouabain can be combined with it to regulate cell proliferation and metabolism. Studying the effect of ouabain on the osteogenic differentiation of bone marrow mesenchymal stem cells can provide ideas for the treatment of bone defects and bone diseases. OBJECTIVE: To explore the effects of different concentrations of Na+/K+-ATPase ligand ouabain on the osteogenic differentiation of human bone marrow mesenchymal stem cells. METHODS: Human bone marrow mesenchymal stem cells were isolated from fresh bone marrow samples from patients that experienced hip replacement, using the whole bone marrow direct adherence method, as experimental cells for inducing osteogenic differentiation. Before osteoinduction, human bone marrow mesenchymal stem cells were treated in different concentrations of ouabain (10, 100, and 1 000 nmol/L) for 24 hours. The untreated human bone marrow mesenchymal stem cells were used as the control group to induce directly. At 21 days after osteoinduction, alizarin red staining was performed. The proportion area was measured using Image Pro Plus software to compare the differences in the amount of calcified nodules produced in each group.  RESULTS AND CONCLUSION: Compared with the control group, as human bone marrow mesenchymal stem cells in vitro treated with ouabain in low concentration (10 nmol/L), the number of calcified nodules increased after alizarin red staining and the staining areas were higher than that in the control group (P < 0.05). The number of calcified nodules observed after treatment in the high concentration group (100, 1 000 nmol/L) was reduced, and the measured stained area was smaller than that in the control group (P < 0.05). The higher the concentration, the fewer the calcifiednodules were. It is concluded that the fresh bone marrow form hip arthroplasty can be used as a test material for isolating human bone marrow mesenchymal stem cells. Low concentrations of ouabain promote differentiation of human bone marrow mesenchymal stem cells into osteoblasts, while a high concentration of ouabain showed an inhibitory effect.  Figures and Tables | References | Related Articles | Metrics

Select

Bone marrow mesenchymal stem cells may alleviate brain damage caused by the microglial overactivation in the cortex around ischemic site of stroke Yan Nan, Wu Yanlong, Tang Xiaohui, Zhang Xiaoyan, Wang Hui, Yang Tianze, Zhou Maochun, Wang Zhengdong, Yang Xiaoxia 2022, 26 (24):  3790-3795.  doi: 10.12307/2022.555 Abstract ( 564 )   PDF (1754KB) ( 136 )   Save BACKGROUND: Bone marrow mesenchymal stem cell transplantation has achieved good results in the treatment of ischemic stroke, but its mechanism of action still needs to be studied in depth.   OBJECTIVE: To study the effects of bone marrow mesenchymal stem cells on further damage of overactivated microglia in the cortex around ischemic site.  METHODS: Thirty 8-week-old Sprague-Dawley rats were divided into sham operation, model, and transplantation groups. The ischemic stroke models of rats were established with blocking the middle cerebral artery with an embolic thread method for the model group and transplantation group. The third generation of bone marrow mesenchymal stem cells was injected into the abdominal cavity of the transplantation. Rabbits of the model group and the sham operation group were injected with the same amount of DMEM. The injection was taken on every other day after surgery for 14 consecutive days. The motor function of paralyzed rats in different groups was evaluated by BBB scores at 7 and 14 days after surgery. At 15 days after operation, the cortex around the cerebral infarction was taken from the rats. Immunohistochemical staining and western blot assay were applied to study the expression of Ox-42, a marker of activated microglia, and tumor necrosis factor-α, interleukin-6, and interleukin-1β in the cortex near the ischemia cortex.  RESULTS AND CONCLUSION: Compared with the sham operation group, expression levels of Ox-42 activated by microglia and tumor necrosis factor-α, interleukin-6, and interleukin-1β were higher in the model group, and decreased in the transplantation group. Compared with the model group, BBB scores showed an improved motor function of paralysis limbs in the transplantation group. It is concluded that the over-activation of microglia is inhibited, and the expression of inflammatory cytokines is decreased with bone marrow mesenchymal stem cells, thus to protect the cerebral cortex from damaging and promote motor function recovery to a certain extent. Figures and Tables | References | Related Articles | Metrics

Select

Effect of paired box gene 6 protein on the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells Wu Shuang, Zou Xingxing, Gao Jie, Hu Rong, Su Min 2022, 26 (24):  3858-3864.  doi: 10.12307/2022.566 Abstract ( 438 )   PDF (3441KB) ( 138 )   Save BACKGROUND: Keratopathy transplantation is faced with many problems, such as serious shortage of corneal donors, postoperative immune rejection and so on. While vigorously advocating corneal donation, there is an urgent need to find a new alternative treatment.   OBJECTIVE: The effect of paired box gene 6 on the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells was investigated by optimizing the culture conditions of bone marrow mesenchymal stem cells. METHODS:  Bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells overexpressing paired box gene 6 were identified according to cell morphology, osteogenic adipogenic differentiation ability, and cell surface antigen expression. According to different cultured cells and culture medium, they were divided into control group (bone marrow mesenchymal stem cells + complete medium), and experimental group A (bone marrow mesenchymal stem cells + conditioned medium), experimental group B (bone marrow mesenchymal stem cells + conditioned medium + cytokine), and experimental group C (bone marrow mesenchymal stem cells overexpressing paired box gene 6 + conditioned medium + cytokine). The expression levels of p63, k14 and k12 were detected by immunofluorescence and flow cytometry at 5 and 10 days after induction. The expression of Ki-67, an indicator of cell proliferation, and the rate of apoptosis, were measured by flow cytometry. Western blot assay and RT-PCR were used to determine the protein and mRNA expression of paired box gene 6.   RESULTS AND CONCLUSION: (1) Before induction and culture, the two groups of cells had the characteristics of bone marrow mesenchymal stem cells. (2) After conditioned culture, spherical or oval cells appeared in the four groups after induction, which showed suspension growth. The expression of keratolimbal stem cell-like cells molecules p63 and k14 was positive in the four groups. The fluorescence expression in the experimental group was significantly higher than that in the control group (P < 0.05), but the expression of corneal epithelial molecule k12 was negative. With the optimization of the culture conditions, the cell proliferation of experimental group C was more obvious, and there were fewer apoptotic cells. After 5 days of induction, the expression of paired box gene 6 protein and mRNA in the experimental groups increased progressively at 5 days after induction. (3) Results suggested that conditioned medium combined with cytokine induction can accelerate the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells. Bone marrow mesenchymal stem cells overexpressing paired box gene 6 are more superior in the speed and proportion of directional differentiation into keratolimbal stem cell-like cells, and it can promote cell proliferation and inhibit cell apoptosis. Figures and Tables | References | Related Articles | Metrics

Select

A new method for extracting human bone marrow mesenchymal stem cells and the comparison with traditional methods Li Shidan, Xing Wei, Xie Xiaoyu, Li Youbin, Wang Shaochuan, Fei Jun 2022, 26 (24):  3814-3820.  doi: 10.12307/2022.559 Abstract ( 1067 )   PDF (2952KB) ( 1440 )   Save BACKGROUND: The methods to extract human bone marrow mesenchymal stem cells, including whole bone marrow adherent culture, density gradient centrifugation, flow cytometry separation and immunomagnetic bead separation. Flow cytometry separation and magnetic bead separation are restricted on equipment and reagents, so they are limited to applied. The whole bone marrow adherent culture method and density gradient centrifugation method are commonly used; however, there are still some shortcomings, such as slow cell growth rate and long centrifugation time. Thus, the efficiency of the experiment will be further improved if a more convenient and short time extraction method of bone marrow mesenchymal stem cells is found.  OBJECTIVE: To introduce a new method of extracting human bone marrow mesenchymal stem cells and compare the extracted cells with those extracted by traditional methods to prove the feasibility and practicability of the new method.   METHODS: Phosphate buffered saline was mixed with human bone marrow tissue at the same ratio, and the mixture was stratified by centrifugation. Human bone marrow mesenchymal stem cells were extracted from the white membrane layer followed by comparison with the cells extracted using whole bone marrow adherent method. The extracted cells were identified as bone marrow mesenchymal stem cells by flow cytometry and multidirectional differentiation potential. The number of primary cells, cell morphology, size, cytoskeleton, expression of surface markers, multidirectional differentiation potential, colony formation, proliferation, migration, and osteogenic differentiation were analyzed by cell counting, crystal violet staining, cytoskeleton staining, scratch test, Transwell migration test, and RT-PCR.  RESULTS AND CONCLUSION: (1) After centrifugation, human bone marrow samples were stratified using phosphate buffered saline to extract human bone marrow mesenchymal stem cells. The number of primary cells extracted by phosphate buffered saline method was more than that of whole bone marrow adherent method. The floating adipose tissue was less in the culture system, and the primary cells grew faster. (2) The cells extracted by phosphate buffered saline and whole bone marrow adhesion were fusiform and expressed CD90, CD105, and CD73, but did not express CD45, CD34, CD11b, CD19, and HLA-DR. They had multidirectional differentiation potential and met the standard of mesenchymal stem cells. (3) There was no significant difference in morphology, size and cytoskeleton of the cells extracted using the two methods. Both groups of cells could form colonies and had migration ability. After osteogenic differentiation, both groups of cells expressed osteoblast markers. (4) The results show that it is feasible and practical to extract human bone marrow mesenchymal stem cells with phosphate buffered saline under certain conditions.  Figures and Tables | References | Related Articles | Metrics

Select

Co-culture of fibroblasts and vascular endothelial cells affects proliferation and osteogenesis of adipose stem cells Zhong Ruiying, Wang Fuke, Yang Guiran, Wang Guoliang, Hou Jianfei, Liao Xinyu 2022, 26 (24):  3833-3839.  doi: 10.12307/2022.562 Abstract ( 572 )   PDF (3194KB) ( 435 )   Save BACKGROUND: The development of tissue engineering has provided a new method for the clinical treatment of bone defects, but the problems of slow formation and slow vascularization of tissue engineered bone have always existed. Previous studies have shown that the combined culture system of vascular endothelial cells and adipose stem cells has better ability to repair bone defects than the single type of cells. Fibroblasts have excellent proliferation characteristics, the ability to secrete and synthesize collagen, contain a variety of regulatory factors, can differentiate into osteogenesis, and have the potential to become excellent seed cells to participate in the construction of tissue engineered bone. OBJECTIVE: To investigate the effects of combined culture of fibroblasts, vascular endothelial cells, and adipose stem cells on the proliferation and osteogenic differentiation of adipose stem cells. METHODS:  (1) The cells were divided into four groups: adipose stem cell group, adipose stem cell + vascular endothelial cell co-culture group, adipose stem cell + fibroblast co-culture group, adipose stem cell + vascular endothelial cell + fibroblast co-culture group. The morphological changes of the cells were observed under an inverted microscope. (2) At 1, 3, 5, 7, and 9 days of co-culture, the proliferation of adipose stem cells in each group was detected by CCK-8 assay and the growth curve was plotted. (3) Alizarin red staining and alkaline phosphatase staining were performed on adipose stem cells in each group at 7, 14, 21, and 28 days of co-culture. (4) At 3 weeks of co-culture, the expression level of bone morphogenetic protein 2 in adipose stem cells in each group was detected by western blot assay.   RESULTS AND CONCLUSION: (1) After 14 days of culture, in the adipose stem cell + vascular endothelial cell + fibroblast co-culture group, some cells fused into clumps and distributed in nests, while in the adipose stem cell group, the cell morphology was single and no cell clusters were found. (2) The morphology of the cell growth curve was basically the same in each group, and the absorbance value increased gradually. The absorbance value of the adipose stem cell + vascular endothelial cell + fibroblast co-culture group was highest, followed by the adipose stem cell + vascular endothelial cell co-culture group, followed by the adipose stem cell + fibroblast co-culture group. (3) At 28 days of co-culture, all the cells in each group showed red positive cells, and the most cells in the adipose stem cell + vascular endothelial cell + fibroblast co-culture group showed red focus. The adipose stem cell group was the least. At 28 days of co-culture, each cell had red positive particles. The number of particles was most in the adipose stem cell + vascular endothelial cell + fibroblast group and adipose stem cell + fibroblast co-culture group, and that in the adipose stem cell group was least. (4) Bone morphogenetic protein 2 was expressed in all cells of each group, and it was more obvious in the adipose stem cell + fibroblast co-culture group and the adipose stem cell + vascular endothelial cell + fibroblast co-culture group. (5) Results confirmed that under the condition of co-culture in vitro, fibroblasts promoted osteogenic differentiation of adipose stem cells, which is more strongly than vascular endothelial cells, but the promotion of proliferation was not as good as that of vascular endothelial cells. The co-culture system of fibroblasts combined with vascular endothelial cells and adipose stem cells has the strongest ability to promote the proliferation of adipose stem cells and the rapid and efficient differentiation into osteoblasts. Figures and Tables | References | Related Articles | Metrics

Select

Epithelial differentiation of adipose stem cells induced by saliva in vitro Li Xiaowen, Xu Yingjie, Jia Mengying, Shi Wei, Jia Xinyu, Gong Zhongcheng 2022, 26 (24):  3865-3869.  doi: 10.12307/2022.567 Abstract ( 491 )   PDF (2407KB) ( 217 )   Save BACKGROUND: Buccal fat pads are often used to repair oral and maxillofacial defects, which can rapidly differentiate the oral mucosa from the epithelium. Because the defect wound is exposed to saliva, it is believed that saliva may promote the epithelialization of adipose stem cells in the buccal fat pad.   OBJECTIVE: To investigate the feasibility of promoting epithelial differentiation of rabbit adipose stem cells by induction of saliva in vitro. METHODS:  Fat harvested from 2-3 months New Zealand white rabbit was isolated, cultured, generated adipose stem cells for passage. Passage 3 adipose stem cells were divided into three groups: negative control group, epidermal growth factor group and saliva group. Adipose stem cells were induced by 10 μg/L epidermal growth factor and 15% saliva in the epidermal growth factor group and saliva group. Cell morphology changes were observed under inverted microscope. The expression of cytokeratin 19 was detected by immunofluorescence and RT-PCR after 7 and 14 days of induction.   RESULTS AND CONCLUSION: (1) After saliva induction, the morphology of adipose stem cells was gradually changed from long spindle shape to short spindle shape or even irregular oblate shape. (2) At 7 and 14 days, cytokeratin 19 was positively expressed in the epidermal growth factor and saliva groups, which was higher than that in the negative control group. The expression level of cytokeratin 19 in the 14-day induction group was significantly higher than that in the 7-day induction group. (3) The expression of cytokeratin 19 mRNA in epidermal growth factor and saliva groups was higher than that in negative control group (P < 0.01). The expression level at 14 days was significantly higher than that at 7 days (P < 0.01). (4) Results suggest that saliva can promote the epithelial differentiation of adipose stem cells to some extent. Figures and Tables | References | Related Articles | Metrics

Select

Effect of adipose-derived stem cell transplantation on the immune microenvironment of adipose tissue in aged mice Wu Siyi, Wu Qiong 2022, 26 (24):  3773-3778.  doi: 10.12307/2022.552 Abstract ( 479 )   PDF (1331KB) ( 90 )   Save BACKGROUND: With the wide application of adipose-derived stem cells, exploring the regulatory mechanism of adipose stem cells is helpful to broaden the choice of materials. OBJECTIVE: To investigate the effect of adipose-derived stem cell transplantation on the immune microenvironment of adipose tissue in aged mice and its mechanism of action in aging-associated obesity.  METHODS: Mouse adipose-derived stem cells were isolated and purified, and transplanted into aged mice at the age of 18 months by intraperitoneal injection. The 18-month-old mice with the same amount of PBS intraperitoneally were used as the old control group, and the 2-month-old mice with the same amount of PBS intraperitoneally were used as the young control group. At 4 weeks after transplantation, changes in food intake and body weight were recorded in each group, and changes in immune cell abundance in adipose, spleen, bone marrow and peripheral blood were analyzed by flow cytometry. The levels of interleukin-6 and tumor necrosis factor-α in adipose tissue and serum were detected using qRT-PCR and ELISA.   RESULTS AND CONCLUSION: (1) The body weight of adipose-derived stem cells-transplanted aged mice decreased by (1.94±0.26) g compared with aged controls. (2) The results of flow cytometry abundance analysis showed that the abundance of myeloid-derived suppressor cells decreased, the abundance of T cells increased, and the abundance of B cells and NK cells tended to develop in adipose-derived stem cells-transplanted aged mice toward young controls. (3) The results of qRT-PCR and ELISA experiments showed that expression levels of interleukin-6 and tumor necrosis factor-α were significantly down-regulated in the adipose tissue and serum of adipose-derived stem cells-transplanted aged mice. (4) In summary, adipose-derived stem cell transplantation was able to suppress the development of obesity in inflammatory aging mice through the modulation of the immune microenvironment in aged mice. Figures and Tables | Related Articles | Metrics

Select

Viability of different fat derivatives in vitro and the outcome after transplantation Zhang Huabin, Zhang Muchen, Liu Chang, Xu Manman, Yin Qichuan, Zhang Aijun 2022, 26 (24):  3779-3784.  doi: 10.12307/2022.553 Abstract ( 515 )   PDF (2786KB) ( 159 )   Save BACKGROUND: Autologous fat is an ideal filler that repairs soft tissue defects, but the long-term survival rate of adipose tissue after transplantation has uncertainty. We explored the effect of adipose tissue structure on the survival of fat transplantation, providing theoretical basis for improving the clinical effect of fat transplantation.   OBJECTIVE: To investigate the viability of different fat derivatives in vitro and the outcome after transplantation. METHODS:  The adipose tissue obtained after liposuction was prepared into four kinds of adipose derivatives: large granular adipose tissue, medium granular adipose tissue, small granular adipose tissue and stromal vascular fraction gel. Fat acquisition rate and histological structural integrity, content, activity and migration ability of adipose-derived stem cells were compared. Four groups of adipose derivatives were randomly injected into the back of nude mice. The survival rate of the grafts, tissue structure observation and CD31 immunohistochemical staining were performed 1 and 12 weeks after transplantation.   RESULTS AND CONCLUSION: (1) The volume acquisition rates of large, medium, small granular fat and stromal vascular fraction gel were (71.43±2.87)%, (57.14±3.11)%, (45.72±3.97)%, and (11.30±2.33)%, respectively. The fat histological structure of the large particles was the most complete, followed by the medium and small granular fat groups, and the tissue structure of stromal vascular fraction gel was basically completely destroyed. (2) The contents of adipose stem cells obtained from large, medium, small granular fat and stromal vascular fraction gel were (5.03±0.56)×107 L-1, (3.77±0.46)×107 L-1, (2.01±0.64)×107 L-1, and (14.84±1.09)×107 L-1, respectively, with significant differences (P < 0.05). (3) The proliferation ability of adipose stem cells in large granular fat group was the best, followed by medium and small granular fat groups; the proliferation ability of stromal vascular fraction gel group was the worst. (4) The migration ability of adipose stem cells in large granular fat group was the best, followed by medium granular fat group, while that in the small granular fat group and stromal vascular fraction gel group was poor. (5) After transplantation, the volume retention rate of stromal vascular fraction gel group was the highest, followed by large granular fat group, while small granular fat group was the worst. (6) After 12 weeks of fat transplantation, fat cells in large granular fat group were partially destroyed; cells in medium granular fat group and small granular fat group were largely destroyed and some vesicles were formed; a large number of mature fat cells were formed in stromal vascular fraction gel group. (7) At 12 weeks after transplantation, the formation of blood vessels in large granular fat group and stromal vascular fraction gel group was more than that in medium granular fat group, while small granular fat group was the least, with significant differences (P < 0.05). There was no significant difference between the large granular fat group and stromal vascular fraction gel group (P > 0.05). (8) Results show that structural integrity of adipose tissue affects adipose stem cell production, vitality and adipose tissue survival rate after transplantation. Suitable options are made according to the characteristics of different fat derivatives to achieve the best filling effect. Figures and Tables | References | Related Articles | Metrics

Select

Isolation, identification, comparison of the differentiation ability of multilineage-differentiating stress-enduring cells from human umbilical cord derived and adipose derived mesenchymal stem cells Zhang Kun, Li Fang, Xiao Dongjie, Wang Yunshan, Huang Guobao, Liu hua 2022, 26 (24):  3802-3807.  doi: 10.12307/2022.557 Abstract ( 628 )   PDF (2339KB) ( 133 )   Save BACKGROUND: Multilineage-differentiating stress-enduring (Muse) cells possess self-renewal and multiple differentiation potential. Muse cells are able to differentiate into all three germ layers, and exhibit non-tumorigenicity and low telomerase activity. It is a seed cell in the fields of tissue engineering, cell transplantation and gene therapy, providing a broad prospect for the treatment of various clinical diseases.  OBJECTIVE: To study the biological characteristics of Muse cells from umbilical cord derived and adipose derived mesenchymal stem cells in vitro, and provide experimental data for obtaining sufficient Muse cells for clinical research.  METHODS: Umbilical cord derived and adipose derived mesenchymal stem cells were incubated with trypsin for different time. The cell survival rates and SSEA-3 expression were detected by Trypan Blue staining and Flow cytomertry. Muse cells were obtained by suspension adherence method. Alkaline phosphatase staining was used to obtain the pluripotency of Muse cells. Muse cells underwent multi-lineage differentiation under different induction conditions. Oil red O staining, Alizarin Red S staining, and Von Kossa staining were used to compare the adipogenic and osteogenic differentiation. RT-PCR was used to detect the changes in pluripotent stem cell marker genes Oct-3/4, Nanog, SSEA-3 and Muse cells induced differentiation related genes doublecortin, neuron-specific enolase, lipoprotein lipase, osteopontin, and alpha-fetoprotein expression.  RESULTS AND CONCLUSION: (1) Under trypsin incubation extension, survival rates of umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells were decreased, accompanied with SSEA-3 expression increased. (2) Alkaline phosphatase staining was positive in Muse cells and negative in mesenchymal stem cells. (3) SSEA-3, Nanog, and Oct-3/4 were highly expressed in Muse cells compared with mesenchymal stem cells. Nanog and Oct-3/4 were highly expressed in adipose-Muse cells compared with umbilical cord-Muse cells. Oil red O, Alizarin Red S, and Von Kossa staining results showed that Muse cells had stronger adipogenetic and osteogenetic abilities than mesenchymal stem cells. (4) Mesenchymal stem cells and Muse cells had different differentiation efficiencies under the action of adipogenesis, osteogenic induction culture medium, neuronal cell induction liquid, and hepatocyte induction liquid, but they all highly expressed lipoprotein lipase, osteopontin, neuron-specific enolase, doublecortin, and alpha-fetoprotein. The above genes were highly expressed in Muse cells compared with mesenchymal stem cells. (5) To conclude, umbilical cord derived and adipose derived mesenchymal stem cells showed the best SSEA-3 positive rate and survival rate after trypsin digestion for 4 hours. The SSEA-3 positive rate of adipose derived mesenchymal stem cells was higher than that of umbilical cord derived mesenchymal stem cells. Muse cells obtained from umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells highly express pluripotent stem cell genes and are able to differentiate into three germ layers.  Figures and Tables | References | Related Articles | Metrics

Select

Effect of umbilical cord mesenchymal stem cells on aging thymus epithelial cells Yang Zailing, Tian Chuan, Lyu Guanke, Pan Hang, Zhu Xiangqing, Wang Jinxiang, Wang Kai, Ruan Guangping, He Zhixu, Shu Liping, Pan Xinghua 2022, 26 (24):  3880-3885.  doi: 10.12307/2022.570 Abstract ( 548 )   PDF (3597KB) ( 150 )   Save BACKGROUND: Previous studies have found that the reduced number and dysfunction of thymus epithelial cells are one of the important factors of thymus degeneration. It can lead to thymic cell development, defective proliferation and peripheral T cell dysfunction. The previous research of our team found that mesenchymal stem cells can reverse the structure and function of aging thymus in vivo. The specific mechanism of action is still unclear.  OBJECTIVE: To investigate the effect of umbilical cord mesenchymal stem cells on aging thymus epithelial cells in vitro. METHODS: The cell viability of the fourth generation thymus epithelial cells was determined by CCK8 assay after treatment with 200 μmol/L H2O2 for 24, 48, 72, and 96 hours, respectively. The 72 hours of 200 μmol/L H2O2 was selected as appropriate time for establishing senescence models. The fourth generation of umbilical cord mesenchymal stem cells was co-cultured with senescent thymic epithelial cells for 48 hours. The changes of senescent cells were detected by β-galactosidase staining. Cell proliferation was detected by immunofluorescence ki-67 staining. Cell cycle changes were analyzed by flow cytometry. Immunohistochemical staining was used to detect the positive expression levels of p53 and p21 proteins. The mRNA expression levels of senescence related genes p21, p53, and pRb were detected by RT-PCR. RESULTS and CONCLUSION: Compared with the model group, the expression of β-galactosidase in coculture group was decreased; cell proliferation ability was increased; cells in G2 phase were decreased and cells in S phase were increased; immunofluorescence expression of p53 and p21 was increased; mRNA expression of p53 and p21 was decreased and pRb mRNA expression was increased. The results of β-galactosidase expression, cell proliferation ability, p53, p21, and pRb expression in the coculture group were compared with the control group, and the reverse degree was close to the control group. It is concluded that umbilical cord mesenchymal stem cells can regulate the senescence of thymic epithelial cells, promote the proliferation and division of thymic epithelial cells, and reverse the senescence of thymic epithelial cells. Figures and Tables | References | Related Articles | Metrics

Select

Osteogenic differentiation of human periodontal ligament stem cells under estrogen and crosstalk between the two pathways Jin Ke, Wu Xiaoling, Luo Xiaoling, Xu Pengfei, Xu Xiaomei 2022, 26 (24):  3886-3891.  doi: 10.12307/2022.571 Abstract ( 529 )   PDF (3348KB) ( 177 )   Save BACKGROUND: Periodontal tissue regeneration engineering based on stem cells provides a broad prospect for the treatment of periodontal diseases. Estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells is closely related to the Wnt signaling pathway. OBJECTIVE: To study the regulation of Wnt/β-catenin and Wnt/Ca2+ signaling pathways on estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells, as well as the crosstalk between the two pathways.   METHODS: Premolars with healthy periodontal condition were collected to isolate and culture human periodontal ligament stem cells. Surface markers of the cells were detected by flow cytometry. The osteogenic/adipogenic differentiation potential was detected by Alizarin red S and Oil red O staining. The third-generation human periodontal ligament stem cells were randomly divided into blank group, control group, estrogen group, XAV939 (Wnt/β-catenin pathway inhibitor) group, L-690,330 (Wnt/Ca2+ pathway inhibitor) group, and XAV939+L-690,330 group. Except for the blank group, all other groups were cultured for osteogenic induction. qRT-PCR and western blot assay were performed to measure expression of osteogenic markers (Runx2, OCN) and key signal molecules of Wnt/β-catenin and Wnt/Ca2+ pathway. Intracellular Ca2+ concentration was evaluated by Fluo-4 fluorescent probe. Alkaline phosphatase assay was conducted to measure the alkaline phosphatase activity. Osteogenic differentiation potential was detected by Alizarin red S staining.  RESULTS AND CONCLUSION: (1) The human periodontal ligament stem cells isolated and purified by collagenase digestion were spindle shaped and polygonal. Flow cytometry proved that human periodontal ligament stem cells were of mesenchymal origin, and they also demonstrated the capacity to differentiate into osteogenic/adipogenic lineages. (2) Estrogen at 10-7 mol/L in the osteogenic medium can upregulate intracellular Ca2+ concentration, CaMKII and NLK gene expression and CaMKII protein expression of human periodontal ligament stem cells. (3) The usage of Wnt/β-catenin and Wnt/Ca2+ signaling pathway inhibitors can minimize osteogenic differentiation. (4) Inhibiting either of the two signaling pathways can increase crucial protein expression of the other pathway, which had no significant effect on the osteogenic differentiation of the cells. (5) In conclusion, estrogen through Wnt/Ca2+ and Wnt/β-catenin signaling pathways induces osteogenic differentiation of human periodontal ligament stem cells. In addition, there is mutual compensation between the two pathways, thus working together to regulate osteogenic differentiation.   Figures and Tables | References | Related Articles | Metrics

Select

In vitro differentiation of human placenta-derived mesenchymal stem cells into hepatocytes Liang Tingting, Chen Li, Fan Mingsong, Zhang Guoying, Zhang Shichang 2022, 26 (24):  3870-3874.  doi: 10.12307/2022.568 Abstract ( 560 )   PDF (4330KB) ( 225 )   Save BACKGROUND: Mesenchymal stem cells are an attractive source of therapies for liver failure. The differentiation of mesenchymal stem cells into hepatocytes can improve the therapeutic effect of liver failure.   OBJECTIVE: To investigate the feasibility of placenta derived mesenchymal stem cells to differentiate into mature hepatocytes in vitro for the treatment of liver failure. METHODS:  Placenta-derived mesenchymal stem cells were isolated and cultured from human placenta tissue. The surface markers and differentiation potential of the isolated cells were identified by flow cytometry, and multidirectional induction. Placenta-derived mesenchymal stem cells were induced into hepatocytes by several cytokines for 22 days. Hepatic markers of induced cells were detected by immunofluorescence staining. Glycogen staining was used to detect the glycogen synthesis ability of cells.   RESULTS AND CONCLUSION: (1) Under the inverted microscopy, the cultured cells were spindle or fibroblast-like cells, which proliferated rapidly. There was no obvious change in cell morphology after 10 passages. (2) Flow cytometry results revealed that the positive rate of CD73, CD90, CD105, CD44, CD166, CD29, CD54, and HLA-ABC in placenta-derived mesenchymal stem cells was more than 95%, while CD34, CD45, CD117, CD14, CD19, CD133, CD31, and HLA-DR were all negatively expressed. (3) The results of oil red O, alizarin red, and alcian blue staining were positive after adipogenic, osteogenic and chondrogenic differentiation of placenta-derived mesenchymal stem cells. (4) After 22 days of hepatic differentiation, these cells exhibited typical morphology of hepatocytes such as round or polygonal shape with obvious cell boundary. Immunofluorescence staining showed that a large number of differentiated cells expressed albumin, CK-18, HepPar1, and CYP7A1 after induction. The positive staining of differentiated cells was observed by glycogen staining. (5) Our results demonstrated that the cells isolated from placenta were mesenchymal stem cells, and placenta-derived mesenchymal stem cells can differentiate into mature hepatocytes in vitro, which could provide reliable sources of hepatocytes for the treatment of liver failure. Figures and Tables | References | Related Articles | Metrics

Select

Effects of mechanical stretch on fiber proliferation in human lung epithelial cells Zhang Rong, Liang Zhenting, Yang Chun, Liu Dongdong, Xi Yin, Zhang Jie, Wang Ya, Xu Yonghao, Liu Xiaoqing, Li Yimin 2022, 26 (24):  3821-3825.  doi: 10.12307/2022.560 Abstract ( 563 )   PDF (1328KB) ( 193 )   Save BACKGROUND: Mechanical ventilation is currently one of important and effective treatment and support methods for severe respiratory diseases, but improper mechanical ventilation can aggravate lung injury and even form fibrosis. How to minimize the lung damage associated with the ventilator and play real protective role of the ventilator is very important. The lung is a mechanical organ. Mechanical ventilation causes the alveoli to be inflated repeatedly, which stretches the neighboring lung epithelial cells, and puts it in an abnormal damage repair. Therefore, excessive mechanical stretch may play an important role in promoting the occurrence of lung fibrosis. At present, there are few reports on the effect of mechanical stretch on the proliferation of lung epithelial cells at home and abroad, and the specific mechanism is not clear. OBJECTIVE: To investigate the effect of different mechanical stretch strengths and stretch time on expression of transforming growth factor β1, vimentin, and type I collagen in human lung epithelial BEAS-2B cells. METHODS: Human lung epithelial BEAS-2B cells were cultured in vitro. Cells at logarithmic growth phase were divided into control static group, 10% stretch group, and 20% stretch group. The FX-5000 system was used to stretch BEAS-2B cells at 20 times/min and sine wave for 24, 48, and 72 hours. The morphological changes in cells were observed with the inverted microscope. The expression levels of transforming growth factor β1, vimentin, and type I collagen mRNA were evaluated by RT-qPCR and immunofluorescence.  RESULTS AND CONCLUSION: (1) BEAS-2B cells displayed cobblestone morphology and linked closely in the control static group. The cell morphology changed from cobblestone shape into long spindle, and intercellular space increased obviously after 20% mechanical stretch for 72 hours. (2) The expression of mRNA and protein of transforming growth factor β1, vimentin, and type I collagen in human lung epithelial BEAS-2B cells was up-regulated with increasing stretch force in a time-dependent manner compared with the control static group (P < 0.05). Compared with the control static group, the changes in transforming growth factor β1, vimentin, and type I collagen were not significant in the 10% stretch group. (3) It is concluded that mechanical stretch increases the expression of transforming growth factor β1, vimentin, and type I collagen in human lung epithelial BEAS-2B cells. Excessive mechanical stretch plays an important role in promoting lung fiber proliferation.  Figures and Tables | References | Related Articles | Metrics

Select

Expression of vasohibin-1 and other factors after the co-culture of two kinds of Echinococcus granulosus protoscolex and endothelial progenitor cells Yu Xiaofan, Jiang Huijiao, Tan Xiaowu, Wu Xiangwei 2022, 26 (24):  3892-3896.  doi: 10.12307/2022.572 Abstract ( 498 )   PDF (1141KB) ( 130 )   Save BACKGROUND: Alveolar echinococcosis and cystic echinococcosis grow in different ways in the body. Alveolar echinococcosis is infiltrating growth, which can invade tissues, blood vessels, and bile ducts. Cystic echinococcosis has fibrous outer cyst wall-wrapped lesions, which show compressive growth of tissues. The effect of the two types of hydatidosis on the invasion and formation of blood vessels is still unclear.   OBJECTIVE: To explore the effects of two kinds of Echinococcus granulosus protoscolex on angiogenesis-related factors in endothelial progenitor cells. METHODS:  After extraction, culture, and identification of endothelial progenitor cells from C57BL/6 mouse bone marrow, and extraction of two types of Echinococcus granulosus protoscolex, two kinds of Echinococcus granulosus protoscolex were co-cultured with endothelial progenitor cells for 24, 48, and 60 hours. ELISA was applied to detect the secretion of vascular endothelial growth factor A and stromal cell-derived factor-1α in the supernatant of cell co-culture. Western blot assay was utilized to detect the relative expression of vasohibin-1, vascular endothelial growth factor A, hypoxia-inducible factor 1α, and stromal cell-derived factor-1α protein in  endothelial progenitor cells of each group.   RESULTS AND CONCLUSION: (1) The levels of vascular endothelial growth factor A and stromal cell-derived factor-1α were significantly higher in the alveolar echinococcosis group than those in the cystic echinococcosis group at 60 hours of coculture (P < 0.05). (2) Relative expression levels of vasohibin-1,  hypoxia-inducible factor 1α, and stromal cell-derived factor-1α were higher in the alveolar echinococcosis group than those in the cystic echinococcosis group at 48 and 60 hours of coculture (P < 0.05). (3) The relative expression levels of vascular endothelial growth factor A were not significantly different in the alveolar echinococcosis and cystic echinococcosis groups at 48 and 60 hours of coculture (P > 0.05). (4) It is concluded that alveolar echinococcosis promotes angiogenesis, and cystic echinococcosis has little effect on angiogenesis. Figures and Tables | References | Related Articles | Metrics

Select

Establishment and identification of an immortalized oral cancer-related fibroblast line Chen Sheng, Yang Yan, Ding Liang, Ye Chuanjin, Zhang Lei, Xia Shu, Li Huiling, Huang Xiaofeng 2022, 26 (24):  3808-3813.  doi: 10.12307/2022.558 Abstract ( 685 )   PDF (1931KB) ( 219 )   Save BACKGROUND: Cancer-associated fibroblasts are important cell components that constitute the tumor microenvironment, but they are prone to aging and phenotype loss during the primary culture, which greatly reduces the experimental reproducibility of cancer-associated fibroblasts function-related research.   OBJECTIVE: To establish an immortalized oral cancer-related cancer-associated fibroblast line and provide stable experimental cells for exploring the function of cancer-associated fibroblasts in oral cancer. METHODS:  Enzymatic digestion method was used to isolate and purify cancer-associated fibroblasts from human oral cancer tissues, and the lentivirus packaged with human telomerase reverse transcriptase (hTERT) (pBABE-hygro-hTERT) was used to infect primary cancer-associated fibroblasts; and their morphology and markers were identified; cell proliferation and aging functions were tested. Furthermore, chromosome karyotype analysis and short tandem repeat analysis were performed on primary cancer-associated fibroblasts and immortalized cancer-associated fibroblasts for cell line identification.   RESULTS AND CONCLUSION: (1) After fibroblasts were immortalized, the expression levels of their markers α-SMA, PDGFRβ and FSP-1 were not significantly changed. (2) Even after 15 passages, their proliferation rate was significantly better than that of non-immortalized cells. (3) The results of β-galactosidase staining showed that with the increase of cell passage, compared with non-immortalized fibroblasts, immortalized fibroblasts did not present obvious senescence. (4) The karyotype of immortalized fibroblasts still retained the basic characteristics of normal cells without malignant transformation. (5) Short tandem repeat identification results demonstrated that immortalized fibroblasts were primary cells, and there was no overlap with the information in the known cell database. (6) These findings indicate that an immortalized fibroblast line derived from oral cancer has been successfully established, which provides a further experimental basis for studying the tumor microenvironment of oral cancer. Figures and Tables | References | Related Articles | Metrics

Select

Effects of red deer horn powder extract on the biological behavior of bone marrow derived macrophages Shan Shuai, Dong Yi, Liu Jialin, Han Xiangzhen, He Huiyu 2022, 26 (24):  3840-3845.  doi: 10.12307/2022.563 Abstract ( 502 )   PDF (1978KB) ( 242 )   Save BACKGROUND: Foreign body reactions will lead to the failure of material implantation. In recent years, research on immunity has gradually become a hot spot. It is of great significance to select scaffold materials with good immune properties for the development of bone defect repair.   OBJECTIVE: To investigate the effects of calcinated antler cancellous bone and untreated antler bone on the biological behavior of bone marrow-derived M1 macrophages. METHODS:  Calcinated antler cancellous bone was prepared by physical and chemical methods. The physical and chemical properties of calcinated antler cancellous bone and untreated antler bone were detected by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The extracts of calcinated antler cancellous bone and untreated antler bone were prepared and the contents of Ca, P and Mg in the extracts were determined. Bone marrow-derived macrophages were cultured in vitro and polarized into M1 macrophages under lipopolysaccharide and interferon γ stimulation. These cells were then co-cultured with different extracts in calcinated antler cancellous bone and untreated antler bone groups, while co-cultured with an IMDM medium in control group, followed by cell counting kit-8 detection of cell survival, flow cytometry detection of M1 markers, and qPCR detection of macrophage-related genes.   RESULTS AND CONCLUSION: (1) The contents of Ca, P and Mg were higher in the calcinated antler cancellous bone group than the untreated antler bone group. (2) The main functional groups in these two groups included OH-, PO43-, and CO32 -. (3) There was no significant difference in cell survival rate among the three groups for 1, 3, and 7 days of co-culture. (4) The expression rate of CD16/32 was lower in the calcinated antler cancellous bone group than the control group after 1-day co-culture. (5) The expression levels of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-6 were lower in the calcinated antler cancellous bone group than the control group after 1 and 3 days of co-culture, while there was no significant difference between the untreated antler bone group and the control group. The expression of CD206, a main marker of M2 macrophages, was significantly higher in the calcinated antler cancellous bone group than the control group at 3 days of co-culture, while there was no significant difference between the untreated antler bone group and the control group at 1 and 3 days of co-culture. (6) To conclude, calcinated antler cancellous bone powder extract can reduce the expression of pro-inflammatory cytokines in M1 macrophages and promote their polarization to M2 macrophages. Figures and Tables | References | Related Articles | Metrics

Select

Autophagy-related gene Rubicon deficiency inhibits cellular senescence in the heart He Fan, Lai Shuaiwei, Zhang Shasha, Dong Fan, Amber Naz, Haniya Mazhar, He Lin, Zhu Hongxin 2022, 26 (24):  3897-3902.  doi: 10.12307/2022.573 Abstract ( 684 )   PDF (1701KB) ( 1300 )   Save BACKGROUND: Cardiac aging is the major risk factor for heart diseases. Cellular senescence is the key mechanism of cardiac aging, which plays an important role in age-related heart disease. Our previous work has shown that deletion of autophagy-related gene Uvrag promotes cellular senescence in the heart. Rubicon is an inhibitory interacting partner of Uvrag. However, the function of Rubicon in cellular senescence in the heart remains unknown.  OBJECTIVE: To determine the effect of Rubicon gene on cellular senescence in the heart.  METHODS: Wild type and Rubicon-deficient mice at 15 months of age were utilized. Uvrag-deficient mice were applied as observation objects. Uvrag-deficient mice of the same age were used as positive controls. Fluorescence quantitative PCR was used to detect changes in mRNA expression of senescence-related secreted phenotype-related factors in mouse heart tissue. Hematoxylin-eosin staining, Sirius red staining, and senescence-associated β-galactosidase staining were performed to observe myocardial histology. Western blot assay was conducted to measure the expression of p53 and p16 protein in the mouse heart. The protocols were approved by the Animal Experiment Ethics Committee of Shanghai Jiao Tong University.    RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining and Sirius red staining demonstrated that Rubicon deficiency ameliorated myocardial cell remodeling and cardiac fibrosis during aging. (2) Senescence-associated β-galactosidase staining displayed that Rubicon-deficient mice had significantly fewer senescence-associated β-galactosidase-positive cells in hearts from Rubicon-deficient mice than in control mice (P < 0.05). (3) Fluorescence quantitative PCR results suggested that expression of interleukin 1β, interleukin 6, transforming growth factor β, type III collagen α1 chain, and tissue inhibitor of matrix metalloproteinase 1 mRNA in hearts was significantly decreased in Rubicon-deficient mice than that in control mice (P < 0.05). (4) Western blot assay results showed that p53 protein abundance, a key protein regulating cellular senescence, was significantly lower in hearts from Rubicon-deficient mice than in control mice (P < 0.05). (5) It is indicated that Rubicon deficiency obviously inhibits cellular senescence in the heart and delays cardiac aging. Rubicon is a potential target for the treatment of cardiac aging and age-related heart disease. Figures and Tables | References | Related Articles | Metrics

Select

CD1d-mediated inhibition of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammatory factor expression via signal regulatory protein-alpha Xiang Mingzhi, Yuan Zilin, Wang Gang, Cheng Jie, Diao Bo, Liu Yueping 2022, 26 (24):  3826-3832.  doi: 10.12307/2022.561 Abstract ( 520 )   PDF (2589KB) ( 205 )   Save BACKGROUND: Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory factors have been proven to be associated with a variety of inflammatory diseases, and regulating their expression will provide some insights into the treatment of related diseases. OBJECTIVE: To explore whether CD1d regulates the expression of NLRP3 inflammatory factors so as to verify the underlying mechanism that CD1d regulates the NLRP3 inflammasomes expression via signal regulatory protein-α (SIRPα). METHODS: (1) 293T cells were transfected with Flag-CD1d and HA-SIRPα lentivirus overexpression vectors for co-immunoprecipitation assay to verify the interaction between CD1d and SIRPα. (2) RAW264.7 was transfected with the Flag-CD1d lentiviral overexpression vector. RT-qPCR was used to detect the expression of NLRP3 and other genes. (3) RAW264.7 was transfected with the overexpression vector of HA-SIRPα lentivirus. The gene and protein expression levels of NLRP3  were detected by RT-qPCR and western blot assay, respectively. (4) SIRPα lentivirus RNAi vector was used to transfect RAW264.7. After lipopolysaccharide stimulation, gene and protein expression levels of NLRP3 were detected by RT-qPCR and western blot assay.  RESULTS AND CONCLUSION: (1) Co-immunoprecipitation assay confirmed the interactions between CD1d and SIRPα. (2) After overexpression of CD1d gene, the expression levels of NLRP3, pro-IL-1β and pro-IL-18 gene in the experimental group were significantly lower than those in the normal group (P < 0.05). (3) The expression of NLRP3 in the experimental group was significantly lower than that in the normal group after lipopolysaccharide stimulation (P < 0.05), but there were no differences in the expression of pro-IL-1β and pro-IL-18 between the experimental group and the control group (P > 0.05). The protein expression of p38, P-P38, NLRP3, pro-IL-1β and pro-IL-18 in the experimental group was significantly lower than that in the SIRPα interference group (P < 0.01). (4) The expression of NLRP3, pro-IL-1β, and pro-IL-18 gene in the experimental group was significantly higher than that in the normal group after lipopolysaccharide stimulation (P < 0.05). The protein expression of p38, p-p38, NLRP3, pro-IL-1β, and pro-IL-18 in the experimental group was significantly higher than that in the overexpression SIRPα group (P < 0.01). (5) It is concluded that CD1d forms a complex with SIRPα on the cell membrane, then inhibits the downstream p38MAPK/NF-κB pathway via SIRPα, and finally inhibits the expression of NLRP3 inflammatory factors.  Figures and Tables | References | Related Articles | Metrics

Select

Effect of transient receptor potential vanilloid 4 agonist GSK1016790A on axonal regeneration Qin Xuzhen, Ma Jinjin, Li Meimei, Wang Xingran, Xie Jile, Saijilafu 2022, 26 (24):  3903-3907.  doi: 10.12307/2022.574 Abstract ( 567 )   PDF (1648KB) ( 126 )   Save BACKGROUND: The transient receptor potential vanilloid 4 (TRPV4) agonist GSK1016790A can activate TRPV4 protein and induce Ca2+ influx in a short time, while its expression remains unchanged. However, during culture, the decreased expression of TRPV4 on HeLa cell membrane surface was induced by GSK1016790A. Whether the phenomenon had an effect on neural regeneration is still not clear.  OBJECTIVE: To explore the effect of TRPV4 agonist GSK1016790A on axonal regeneration in different periods. METHODS: The dorsal root ganglion cells of ICR mouse at 6-8 weeks were treated by collagenase and trypsin for cell culture. In the 2-hour group, the agonist GSK1016790A was added at the initial stage of culture and the cells were treated for 2 hours and the culture was continued for 3 days. The 3-day group was treated with the agonist GSK1016790A for 3 days. The blank control group did not undergo any treatment. The TRPV4 protein expression and axon regeneration related protein expression were examined by western blot assay. TUJ1 and TRPV4 immunofluorescence staining was performed simultaneously. Number of axon branches, length of axon regeneration, and number of cell survival were counted.  RESULTS AND CONCLUSION: (1) Compared with the blank control group, there was no significant difference in TRPV4 protein changes after GSK101 stimulated nerve cells for 2 hours (P > 0.05); while the expression of TRPV4 decreased significantly after nerve cells were stimulated for 3 days (P < 0.05). (2) Compared with the blank control group, after GSK1016790A stimulated nerve cells for 2 hours, there was no significant difference in the number of axon branches and axon length (P > 0.05); after stimulating nerve cells for 3 days, the number of axon bifurcations and axon length were increased significantly (P < 0.05). (3) Compared with the blank control group, there was no significant change in cell survival rate after GSK1016790A stimulated nerve cells for 2 hours or 3 days. (4) Compared with the blank control group, after GSK1016790A stimulated nerve cells for 3 days, the expression of PTEN decreased (P < 0.05), and the regeneration of nerve axons was significant. This process may be mediated by inhibiting the expression of PTEN protein. Figures and Tables | References | Related Articles | Metrics

Select

Antibody detection and cross-matching results in patients with ineffective platelet transfusion in Shenyang region Wang Hongyang, Li Xiaofeng, Zhou Zhuren, Li Hanpin, Cong Rijiao, Li Jianping 2022, 26 (24):  3875-3879.  doi: 10.12307/2022.569 Abstract ( 558 )   PDF (1087KB) ( 308 )   Save BACKGROUND: With the widespread use of platelet transfusion, patients with ineffective platelet transfusion are increasing year by year. OBJECTIVE: To provide a reference for improving the effect of platelet transfusion in patients with ineffective platelet transfusion by detecting and analyzing platelet antibody and cross-matching. METHODS: The platelet antibody test kit (solid phase agglutination method) was used to detect platelet antibody and cross-matching test in 112 patients with ineffective platelet transfusion from April to December 2020. RESULTS AND CONCLUSION: (1) The results of platelet antibody test were positive in 103 of 112 cases. The positive rate was as high as 92%. (2) The results of the platelet cross-matching type showed that 25 patients were incompatible with the donor’s platelets in the cross-matching experiment, and 18 patients were matched with all the donor’s platelets. (3) Relationship between platelet antibody test results and matching rate: The results of platelet antibody test were 84% in patients with negative platelet antibody test, 75.4%, 71.6, 54.6%, and 27.6% in patients with 1+, 2+, 3+, and 4+ platelet antibody test. Differences between groups were statistically significant (P < 0.05). (4) Relationship between the positive rate of platelet antibodies and the patients’ multiple examinations: The positive rate of platelet antibody was 90.2% and 94.2% in the number of platelet transfusions < twice and ≥ twice (P > 0.05). (5) Effect of platelet infusion on the patient’s matching rate: In each group with the same number of inspections, there was no statistically significant difference in the matching rate between the initial inspection and repeated inspections (P > 0.05). In all patients with repeated inspections, the average matching rates of the initial inspection and repeated inspections were 41.2% and 50.6%, respectively, and the difference was not statistically significant (P > 0.05). (6) These results suggest that the higher the positive degree of platelet antibody test, the lower the matching rate was. The positive intensity of platelet antibody did not show an increasing trend after infusion of matched platelets. The patient obtained a stable transfusion effect. Accurate detection of platelet antibody and infusion of matched platelets have an important clinical value for improving the efficacy of platelet transfusion. Figures and Tables | References | Related Articles | Metrics

Select

Effect and mechanism of mesenchymal stem cells on aging-related ischemic stroke Chen Na, Wang Xiaohan, Zhang Yunke 2022, 26 (24):  3914-3920.  doi: 10.12307/2022.576 Abstract ( 524 )   PDF (1403KB) ( 188 )   Save BACKGROUND: Aging has an important relationship with the occurrence and development of ischemic stroke. Mesenchymal stem cells have the functions of self-renewal, differentiation and secretion, and may play a certain role in the treatment of ischemic stroke. OBJECTIVE: To review the relationship between aging and ischemic stroke and the mechanism of action of mesenchymal stem cells in the treatment of aging-related ischemic stroke. METHODS: The articles on the relationship between aging and ischemic stroke and mesenchymal stem cells for treatment of ischemic stroke published from 2006 to 2021 were searched in Wanfang, CNKI, PubMed, and Web of Science. The key words were “aging, mesenchymal stem cells (MSCs), ischemic stroke” in Chinese and English. A total of 58 articles were included for analysis.  RESULTS AND CONCLUSION: (1) Mesenchymal stem cells can differentiate and secrete nutritional factors and vesicles and can be used for the treatment of aging-related ischemic stroke. (2) The pathological process of aging includes the accumulation of senescent cells to activate the secretion of inflammatory factors related to aging, which deteriorates the microenvironment of cell survival. The inflammatory response makes neurovascular unit damage after aging even worse. Oxidative stress causes the destruction of intracellular mitochondrial structure and dysfunction. The shortening of grains and changes in genetic material are closely related to aging and affect the stability of atherosclerosis, and are closely related to the occurrence and development of ischemic stroke. (3) The action mechanisms of mesenchymal stem cells in the treatment of aging-related ischemic stroke mainly include: removing senescent cells and blocking the damage of inflammatory mediator pathways, inhibiting the expression of pro-inflammatory factors, reducing inflammation, neurogenesis, promoting nerve function repair, increasing cerebral blood flow, promoting angiogenesis, secreting exosomes and having similar function of mesenchymal stem cells, which can provide relevant reference for mesenchymal stem cell treatment of ischemic stroke in the future. (4) Although mesenchymal stem cell transplantation can play a role in the treatment of ischemic stroke, it needs to be further resolved that the migration rate and distribution after transplantation, the types and distribution of differentiated cells, survival rate, proliferation, brain three-dimensional local injection or arteriovenous injection. Exploring more effective transplantation strategies is a key factor in order to prevent atherosclerosis and stroke by combination with anti-aging drugs in the future. Figures and Tables | References | Related Articles | Metrics

Select

Transcription factor-miRNA-mRNA network analysis of osteogenic differentiation of adipose-derived stem cells Yu Chunbo, Li Dayu, Fan Fang, Li Changfu 2022, 26 (24):  3908-3913.  doi: 10.12307/2022.575 Abstract ( 707 )   PDF (14528KB) ( 107 )   Save BACKGROUND: Adipose stem cells have a wide range of sources and are easy to obtain. The changes in gene expression involved in the entry of human adipose tissue-derived stem cells into differentiated bone cells are mainly regulated by miRNA and transcription factor, but the specific molecular mechanisms that regulate the osteogenic differentiation of adipose stem cells and their potential transcription factor-miRNA-mRNA regulatory network have not yet been established.   OBJECTIVE: To screen the differentially expressed genes, differentially expressed miRNAs and transcription factors during the osteogenic differentiation of adipose-derived stem cells, and to explore the potential target genes, miRNAs, transcription factors, and transcription factor-miRNA-mRNA regulatory network during the osteogenic differentiation of adipose-derived stem cells. METHODS:  GEO database was used to acquire three chips (GSE37329, GSE63754 two miRNA gene chips and GSE72429 one miRNA data chip) to screen differentially expressed genes and differential miRNAs. Differentially expressed genes were analyzed for PPI network, GO function, and KEGG signaling pathway. Differentially expressed genes were utilized to predict upstream miRNAs and differential miRNAs pre-transcription factors. Transcription factor-miRNA-mRNA network diagrams were constructed through Cytoscape 3.7.1 software.   RESULTS AND CONCLUSION: Eighty-six differentially expressed genes (26 up-regulated genes, 60 down-regulated genes) and 16 differential miRNAs (10 up-regulated miRNAs, 6 down-regulated miRNAs) were screened out. There were 76.52% of protein interactions and co-expression and 8.69% of synergistic localization in the PPI network. GO function enrichment analysis was mainly related to the negative regulation of the classical Wnt signaling pathway and the negative regulation of cartilage development. The KEGG signaling pathway involved signaling pathways, such as tyrosine metabolism and fatty acid degradation. Differentially expressed genes predicted 11 381 miRNAs and differential miRNAs predicted 55 transcription factors. Transcription factor-miRNA-mRNA regulatory network was constructed. The key genes in the osteogenic differentiation of adipose-derived stem cells may be PODXL, SEMA3D, ADGRG6, LGR4, LPL, CADM3, GRIA1, GPM6B, RERG, APCDD1, and NRCAM. Important miRNAs were: hsa-miR-762, hsa-miR-502-3p, and hsa-miR-1275. The core transcription factors were CTCF, TAL1, STAT2, STAT1, and TCF3. The results show that the transcription factor-miRNA-mRNA regulatory network constructed through bioinformatics technology and database mining methods provides potential therapeutic targets for the in-depth study of osteogenic differentiation of adipose-derived stem cells. Figures and Tables | References | Related Articles | Metrics

Select

Human umbilical cord mesenchymal stem cells in endometrial injury repair Liu Xingyu, Hu Xiaofang, Xu Guangli, Wang Rui, Li Peiyao, Wang Mengyuan, Peng Li, Zhu Xiangying 2022, 26 (24):  3921-3927.  doi: 10.12307/2022.577 Abstract ( 761 )   PDF (1377KB) ( 259 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells have the characteristics of strong differentiation and immunoregulation, which have been widely concerned in cell therapy. OBJECTIVE: To summarize the research progress of human umbilical cord mesenchymal stem cells in the treatment of endometrial related diseases. METHODS: Key words were “human umbilical cord mesenchymal stem cells, endometrium, endometriosis, intrauterine adhesions, thin endometrium” in Chinese and English. PubMed, CNKI, and Wanfang databases were retrieved for articles published from 1991 to 2020. Totally 64 articles meeting the inclusion criteria were reviewed and analyzed.  RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cells, as a kind of stem cells with considerable development potential, have the advantages of rich sources, convenient collection, significant cell proliferation and differentiation. (2) Human umbilical cord mesenchymal stem cells promote the repair of endometrial cell injury, reduce scar formation and improve endometrial function through immune regulation, cell differentiation and down-regulation of fibrotic gene expression, which is conducive to embryo implantation and pregnancy. (3) The important mechanism of human umbilical cord mesenchymal stem cell transplantation for endometrium may be related to their paracrine cytokines and nutritional factors, and these factors participate in endometrial repair and immune regulation. (4) The treatment method based on human umbilical cord mesenchymal stem cells has achieved some promising results in the treatment of endometrial injury diseases, such as inhibiting the proliferation and promoting the apoptosis of endometrial cells in vitro, and restoring the fertility of endometriosis rats by repairing the damaged endometrium. It can remarkably promote the regenerative repair of endometrium in rats with severe intrauterine adhesion syndrome, promote vascular regeneration, restore endometrial function, increase the thickness of endometrium through immune regulation, improve endometrial receptivity, and provide new ideas for the treatment of thin endometrium. (5) Human umbilical cord mesenchymal stem cells can escape the recognition of the body’s immune system and avoid host monitoring, so infusion into the body will not cause strong rejection. (6) At present, most of the research is still limited to animal experiments, and its application in clinical treatment is not extensive. In the future, more clinical trials are needed to further study treatment mechanism of human umbilical cord mesenchymal stem cells for endometrium. Figures and Tables | References | Related Articles | Metrics

Select

Role and clinical application prospects of exosomal non-coding RNAs in the occurrence and development of glioma Liu Chao, Zeng Zhaomu, Wen Xichao, Wu Wensong, Sun Chengyuan, Zheng Kebin 2022, 26 (24):  3928-3936.  doi: 10.12307/2022.578 Abstract ( 486 )   PDF (1308KB) ( 178 )   Save BACKGROUND: The prognosis of malignant glioma is still poor after surgery, radiotherapy and chemotherapy. Therefore, it is important to clarify the molecular mechanism of glioma development and further explore reliable biomarkers. Non-coding RNAs are enriched and stable in exosomes, and have attracted extensive attention due to their regulatory functions in the initiation and progression of various tumors. OBJECTIVE: To summarize the research and progress of exosomal non-coding RNA in glioma, introduce the biological sources of exosomal non-coding RNA, elucidate its biological effects in glioma, search for reliable tumor markers, and provide new ideas for the diagnosis and treatment of glioma. METHODS: Using “exosome, extracellular vesicles, noncoding RNA, glioma, molecule mechanism, biomarkers” as English search terms, 114 articles were searched and summarized in PubMed and Web of Science databases, mainly involving tumor cell proliferation, invasion and angiogenesis of exosomal non-coding RNA in glioma, as well as gene diagnosis and gene therapy of exosomal non-coding RNA in glioma.  RESULTS AND CONCLUSION: (1) Exosomal non-coding RNA selectively packages, secretes and metastases between cells, participates in intercellular communication in tumor microenvironment, and regulates many biological behaviors of gliomas, such as proliferation, invasion, angiogenesis, immune escape and therapeutic drug resistance, which plays an important role in the occurrence and development of gliomas. (2) Exosomes contain a variety of functional molecules, which can reflect the complex heterogeneity of the tumor. (3) Exosomes have higher specificity and sensitivity, and are very stable, and can be easily obtained in almost all types of body fluids. These factors make exosomes become the most potential biomarkers, which will be of great help for the diagnosis of gliomas in the future. (4) Exosomes are natural and non-toxic, which are quite similar to the structure of plasma membrane. It can be used as a therapeutic carrier for many therapeutic drugs, carry a variety of bioactive molecules and can easily cross the blood-brain barrier. Through exosomes to develop targeted anti-glioma drugs, it can not only break through the blood-brain barrier, but also avoid immune rejection. (5) The combination of exosomes and target cells has specific targeting, which greatly improves the feasibility of biological targeted therapy, but there are still some difficulties in clinical application, such as the difficulty of sorting exosomes, the limited content of exosomal non-coding RNA, and how to make exocrine reach the tumor site accurately. (6) With the further study of exosomal non-coding RNA in glioma, it will provide a new way to overcome the problem of glioma in the future. Figures and Tables | References | Related Articles | Metrics