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    08 January 2020, Volume 24 Issue 1 Previous Issue    Next Issue
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    Effects of vascular endothelial growth factor combined with platelet-derived growth factor-BB on the angiogenic and proliferative abilities of bone marrow mesenchymal stem cells
    Sun An, Bi Xiangyu, Han Xiangzhen, Zhou Qiqi, He Huiyu
    2020, 24 (1):  1-6.  doi: 10.3969/j.issn.2095-4344.2001
    Abstract ( 563 )   PDF (759KB) ( 200 )   Save

    BACKGROUND: Bone tissue engineering provides a new path to treat critical-sized bone defects. However, a stable osteogenesis and osseointegration can only be insured via formation of an intact vascular network, which can achieve satisfactory therapeutic effects. Thereafter, angiogenesis is a challenge and difficulty in bone tissue engineering.

    OBJECTIVE: To investigate the effect of combined application of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB) on proliferation and angiogenesis of bone marrow mesenchymal stem cells.

    METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured and isolated in vitro, and interfered with different concentrations of VEGF (20, 40, 60, 80, 100, 120 μg/L) and PDGF-BB (20, 40, 60, 80, 100, 120 μg/L) in combination or 100 μg/L VEGF, and 100 μg/L PDGF-BB alone. The optimum concentration of promoting cell proliferation was detected by cell counting kit-8 assay. The expression levels of angiopoietin-1, hypoxia-inducible factor-1α, hepatocyte growth factor and insulin-like growth factor were detected by RT-PCR at 7 and 14 days after intervention.

    RESULTS AND CONCLUSION: (1) After growth factors were added, the cell proliferative ability was significantly improved, and combined application revealed better effect. The optimal concentration grou was 80 μg/L VEGF+80 μg/L PDGF-BB. (2) Both VEGF and PDGF-BB could promote the mRNA expression levels of angiopoietin-1, hypoxia-inducible factor-1α, hepatocyte growth factor and insulin-like growth factor, and the effect was more obvious in combined application. (3) The mRNA expression levels of hypoxia-inducible factor-1α and hepatocyte growth factor were significantly increased with time (P < 0.05). The mRNA expression levels of angiopoietin-1 and insulin-like growth factor were significantly decreased with time (P < 0.05). (4) In vitro experimental results suggest that VEGF and PDGF-BB at the concentration of  80 μg/L can consistently promote angiogenesis, and the effect of combined application is better than that of single application.

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    Changes in serum levels of fibroblast growth factor-2, hypoxia-inducible factor-1alpha and vascular endothelial growth factor in patients with avascular necrosis of femoral head treated by enriched autologous bone marrow stem cell transplantation combined with core decompression
    Zhang Jingyi, Hai Guodong, Yang Haofei, Fan Congliang, Yan Lei, Zhang Chunlei, Dou Junfeng, Ma Hailong
    2020, 24 (1):  14-14.  doi: 10.3969/j.issn.2095-4344.2002
    Abstract ( 325 )   PDF (859KB) ( 86 )   Save

    BACKGROUND: Avascular necrosis of the femoral head is mainly caused by insufficient blood supply of the femoral head. Core decompression is a common clinical treatment, enriched autologous bone marrow stem cell transplantation can improve hip function, and combined application can promote early recovery of patients.

    OBJECTIVE: To investigate the therapeutic effects of enriched autologous bone marrow stem cell transplantation combined with core decompression on serum levels of fibroblast growth factor-2, hypoxia-inducible factor-1α and vascular endothelial growth factor in patients with early avascular necrosis of femoral head.

    METHODS: Sixty-two cases of early avascular necrosis of femoral head treated from January 2016 to January 2018 were selected, and were divided into study group (n=31) and control group (n=31) according to the random number table method. The control group was treated with fine-needle porous-channel core pulp decompression, while the study group was treated with enriched autologous bone marrow stem cell transplantation on the basis of the control group. Harris score, Visual Analogue Scale score, serum fibroblast growth factor-2, hypoxia-inducible factor-1α and vascular endothelial growth factor levels and adverse reactions were observed before and after treatment.

    RESULTS AND CONCLUSION: (1) The Harris scores at 3, 6 and 12 months after treatment in the two groups were higher than those before treatment (P < 0.05), and those in the study group were higher than those in the control group (P < 0.05). (2) The Visual Analogue Scale scores at 3, 6 and 12 months after treatment in the two groups were lower than those before treatment (P < 0.05), and the scores at 3 and 6 months after treatment in the study group were lower than those in the control group (P < 0.05). (3) The levels of serum fibroblast growth factor-2 at 3, 6 and 12 months in the two groups were higher than those before treatment, and the scores in the study group were higher than those in the control group (P < 0.05). The levels of hypoxia-inducible factor-1α and vascular endothelial growth factor in the two groups were significantly lower than those before treatment (P < 0.05), and those in the study group were lower than those in the control group (P < 0.05). (4) There was no significant difference in the incidence of adverse reactions between two groups after treatment (P > 0.05). (5) These results suggest that enriched autologous bone marrow stem cell transplantation combined with core decompression in the treatment of early avascular necrosis of femoral head can effectively improve hip joint function, promote the repair of femoral head necrosis area, alleviate pain, increase the level of fibroblast growth factor-2, and reduce the levels of hypoxia-inducible factor-1α and vascular endothelial growth factor.

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    Overexpression of P75 neurotrophin receptor negatively regulates osteogenic differentiation of bone marrow mesenchymal stem cells in vitro
    Zhu Lunjing, Duan Jiangtao, Huang Yijie, Wang Ning, Chen Junyi, Ma Yuegang, Bei Chaoyong
    2020, 24 (1):  20-26.  doi: 10.3969/j.issn.2095-4344.1856
    Abstract ( 493 )   PDF (865KB) ( 188 )   Save

    BACKGROUND: P75 neurotrophin receptor (P75NTR) is widely expressed in nerve tissues and cells, and plays a dual role in promoting or inhibiting differentiation. P75NTR is also overexpressed in local tissues with fracture nonunion. Therefore, P75NTR is studied for the osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance to provide important targets for the clinical treatment of fracture nonunion.

    OBJECTIVE: To elucidate the effect of P75NTR overexpression on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

    METHODS: The bilateral femurs of Sprague-Dawley rats were selected, and the rat bone marrow mesenchymal stem cells were extracted by whole bone marrow separation and adherence method and subcultured in vitro. The P75NTR overexpression plasmid GV358-P75NTR expressing enhanced green fluorescent protein was constructed, and the P75NTR overexpression lentiviral vector was collected by empty lentiviral vector packaging. Rat bone marrow mesenchymal stem cells primary cultured in vitro for 10 days were selected, and seeded into culture plates after digestion. P75NTR overexpression lentivirus and related infection reagents were added for subsequent infection experiments. After 7 days of infection, the expression of green fluorescent protein was observed by fluorescence microscope and overexpression of P75NTR protein was detected by western blot. Transfected cells were cultured for 7 days in a conventional medium, followed by culture in the osteogenic differentiation medium. Alkaline phosphatase activity was quantified by enzyme linked immunosorbent assay on the 7th, 10th, and 14th days after osteogenic induction. Formation of mineralized nodules was observed by alizarin red staining on the 7th and 14th days after osteogenic induction.

    RESULTS AND CONCLUSION: P75NTR overexpression lentiviral vector-infected bone marrow mesenchymal stem cells expressed green fluorescent protein (infection efficiency was about 90%), and the expression of P75NTR protein was significantly increased, which was significantly different from that in the negative control group (P < 0.05). Cell model of P75NTR overexpression was successfully constructed. Compared with the negative control and blank control groups, the alkaline phosphatase activity of the P75NTR overexpression group was significantly decreased at the corresponding time point after osteogenic induction, the number of mineralized nodules was significantly reduced, and cell aggregation and distribution were significantly weakened (P < 0.05). To conclude, P75NTR overexpression negatively regulates the osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured in vitro. Overexpression of P75NTR in local tissues inhibits the osteogenic differentiation of surrounding bone marrow mesenchymal stem cells, which may be an important factor for bone defects or fracture nonunion.


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    Biocompatibility of tissue engineered cartilage constructed in vivo by silk fibroin-chitosan scaffold carrying bone marrow mesenchymal stem cells
    She Rongfeng, Zhang Yi, Chen Long, Wang Yuanzheng, Zhang Bin, Huang Qixiang
    2020, 24 (1):  27-32.  doi: 10.3969/j.issn.2095-4344.1850
    Abstract ( 515 )   PDF (762KB) ( 169 )   Save

    BACKGROUND: Our previous studies have found that silk fibroin-chitosan scaffold carrying bone marrow mesenchymal stem cells can repair cartilage defect in rabbits, but further exploration on the biocompatibility of tissue engineered cartilage is yet to be done. 
    OBJECTIVE: To explore the biocompatibility of tissue engineered cartilage that is constructed in vitro by silk fibroin-chitosan scaffold with bone marrow mesenchymal stem cells.
    METHODS: Three-dimensional silk fibroin-chitosan scaffolds were prepared in a ratio of 1:1. Rabbit bone marrow mesenchymal stem cells were extracted, induced and seeded onto the silk fibroin-chitosan scaffold to construct the cell-scaffold composite. The composite was then implanted into a rabbit joint defect model for cartilage repair. There were three groups in the present study: experiment group with implantation of induced bone marrow mesenchymal stem cells+silk fibroin-chitosan scaffold into the cartilage defect model, control group with implantation of silk fibroin-chitosan scaffold into the cartilage defect model, and blank group without implantation.   
    RESULTS AND CONCLUSION: The three-dimensional silk fibroin-chitosan scaffolds were successfully prepared and combined with bone marrow mesenchymal stem cells (BMSCs) to construct the tissue engineered cartilage for repair cartilage defects in rabbits. Blood routine parameters, procalcitonin levels, erythrocyte sedimentation rates and C-reactive protein levels detected at 2, 4, 8, and 12 weeks post-implantation indicated no obvious signs of systemic infection, and there was no damage to liver and kidney functions in the three groups. There were also no significant differences between the three groups in terms of blood routines and liver and kidney functions (P > 0.05). As shown by gross observation, hematoxylin-eosin staining and scanning electron microscope, in the experimental group, cartilage defects were repaired, with scaffold degradation, no presence of inflammatory cells, and good integration with surrounding tissues. Therefore, tissue engineered cartilage constructed in vitro by silk fibroin-chitosan scaffolds carrying bone marrow mesenchymal stem cells has good biocompatibility, which provides an experimental basis for tissue engineering approaches to cartilage repair.

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    An increase in proliferation of rat bone mesenchymal stem cells and secretion of vascular endothelial growth factor under negative pressure in vitro
    Yang Xiongfeng, Jiang Huijiao, Wang Xiaoyi, Guo Lijiao, Zhou Qing, Han Huanhuan, Li Linlin, Liao Zhenyu, Lei Zhen, Chen Xueling, Zhang Hongwei, Wu Xiangwei
    2020, 24 (1):  33-39.  doi: 10.3969/j.issn.2095-4344.2006
    Abstract ( 395 )   PDF (933KB) ( 150 )   Save

    BACKGROUND: How to enhance the proliferative activity of bone marrow mesenchymal stem cells and make their proper effects after transplantation is an urgent problem to be solved.

    OBJECTIVE: To investigate the effects of different negative pressures on the proliferation of rat bone mesenchymal stem cells and vascular endothelial growth factor secretion level.

    METHODS: Passage 3 bone marrow mesenchymal stem cells were cultured under normal conditions (control group) or under intermittent negative pressures (-6.65, -13.3, -26.6 kPa), once for 2 hours, with an interval of 12 hours. At 12, 24, 36, 48, and 60 hours of culture, cell proliferation was detected using cell counting kit-8, and mRNA expression of vascular endothelial growth factor receptor was detected by RT-PCR. Based on the above results, we selected the optimal negative pressure condition and time (-26.6 kPa, 24 hours). Bone marrow mesenchymal stem cells were treated normally, under -26.6 kPa or treated with Axitinib under -26.6 kPa. We detected the cell proliferation by cell counting kit-8, EDU cell positive rate by EDU kit, and cell colony forming unit by crystal violet staining thereafter.

    RESULTS AND CONCLUSION: Compared with the control group, the negative pressure groups had significantly increased cell proliferation, significantly increased vascular endothelial growth factor levels, and significantly up-regulated mRNA expression of vascular endothelial growth factor receptor (all P < 0.05). After being treated with Axitinib, the absorbance value of cell proliferation, the number of clone forming units and the rate of EDU positive cells in the negative pressure groups were markedly decreased. To conclude, in vitro negative pressure culture may promote the proliferation of mesenchymal stem cells through upregulation of vascular endothelial growth factor expression.


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    Effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cell sheet
    Zhang Wenjing, Wang Jia, Tian Mengting, Han Xiangzhen, Zhou Qiqi, He Huiyu
    2020, 24 (1):  65-71.  doi: 10.3969/j.issn.2095-4344.2009
    Abstract ( 393 )   PDF (1106KB) ( 91 )   Save

    BACKGROUND: The effects and mechanisms of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on the proliferation and osteogenic differentiation of bone mesenchymal stem cells still remain unknown. How to combine the growth factors with tissue-engineered cell patch clamp techniques is of great significance for bone defect repair.

    OBJECTIVE: To explore the effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 applied alone or in combination on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell sheet. 

    METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated, cultured and identified in vitro to construct cell sheet. Bone morphogenetic protein 2 and basic fibroblast growth factor 2 at different concentrations were individually or jointly used to induce bone marrow mesenchymal stem cell sheet. The cell counting kit-8 assay combined with alkaline phosphatase activity assay was used to determine the optimal concentration of the two factors in promoting cell proliferation and osteogenic differentiation. Osteogenic induction of bone marrow mesenchymal stem cell sheet was assessed by gross and microscopic observations, Vonkossa staining, alizarin red staining, and RT-PCR detection. 

    RESULTS AND CONCLUSION: The single application of bone morphogenetic protein 2 enhanced the alkaline phosphatase activity of the bone marrow mesenchymal stem cell sheet, and the optimal concentration was 100 μg/L (P < 0.001). The single application of basic fibroblast growth factor 2 accelerated the proliferation of bone marrow mesenchymal stem cell sheet, and the optimal concentration was 20 μg/L (P < 0.001). Their combination facilitated the proliferation of the cell sheet, and boosted the alkaline phosphatase activities (P < 0.001). The four groups of cell sheet showed no significant morphological difference, and the osteogenic differentiation of the bone marrow mesenchymal stem cell sheet could all be induced through the osteogenic induction. Calcium nodules were most significant in the combination group (< 0.001), suggesting that the combination significantly facilitated late osteogenic differentiation, suppressed early osteogenic differentiation of the sheet and showed significant synergistic effect (P < 0.001). In summary, the application of bone morphogenetic protein 2 combined with basic fibroblast growth factor 2 plays a synergistic role in promoting the proliferation of bone marrow mesenchymal stem cell sheet and significantly enhances the osteogenic induction.

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    Psoralen induces proliferation of rabbit endosteal mesenchymal stem cells and constructs cell-scaffold bone complex in nonunion repair
    Lai Lijin, Mo Haoxuan
    2020, 24 (1):  40-44.  doi: 10.3969/j.issn.2095-4344.2007
    Abstract ( 341 )   PDF (676KB) ( 68 )   Save

    BACKGROUND: Psoralen is a plant estrogen, and a large number of studies have confirmed its role in promoting cell proliferation and differentiation.

    OBJECTIVE: To construct a cell-scaffold composite bone using psoralen, rabbit endosteal mesenchymal stem cells and polycaprolactone and to explore its effect on the treatment of rabbit nonunion.

    METHODS: (1) Rabbit endosteal mesenchymal stem cells were cultured and cultured until the third generation for each experiment. Passage 3 cells were seeded onto culture plates containing 50 mg/L bone morphogenetic protein 2 (positive control), 10-8, 10-7 and 10-6 mol/L psoralen (low-, middle-, and high-concentration psoralen groups) or the same volume of purified water (control group). The cell proliferation of each group was detected on the 3rd, 5th and 7th days after intervention using MTT method. (2) The three-dimensional polycaprolactone scaffold was added to the bottom of the cell culture plate, and rabbit endosteal mesenchymal stem cells were seeded into the scaffold at a density of 1×103 per well. Then, 10-6 mol/L of psoralen was added. Cell-scaffold composite bone was taken after 21 days of culture. (3) Animal models of radial nonunion were established in 27 New Zealand white rabbits, and were then randomized into experimental, scaffold and control groups followed by implantation of cell-scaffold composite bone, simple scaffold, and nothing, respectively. Pathological hematoxylin-eosin staining for observation of bone healing was performed at the 2nd, 4th, and 8th weeks after surgery. Healing of nonunion was observed on the X-ray films that were taken at the 4th week after surgery.

    RESULTS AND CONCLUSION: (1) Three concentrations of psoralen could induce the proliferation of rabbit endosteal mesenchymal stem cells. Compared with the control group, 10-6 mol/L psoralen exerted the strongest stimulation effect on rabbit endosteal mesenchymal stem cells (P < 0.05), whereas there was no significant difference between high-concentration psoralen group positive control group (P > 0.05). (2) Pathological hematoxylin-eosin staining of radial nonunion showed that the number of osteoblasts in the experimental group was higher than that in the scaffold and control groups (P < 0.05). (3) The X-ray films revealed bone healing in the experimental group, partial healing in the scaffold group and non-healing in the control group. Overall findings indicate that psoralen can promote the proliferation of rabbit endostealmesenchymal stem cells, and the effect is certainly related to the concentration of psoralen. Psoralen can be combined with rabbit endosteal mesenchymal stem cells and polycaprolactone scaffold to form composite bone, achieving good outcomes in the treatment of nonunion in animals.

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    Conditioned medium of adipose-derived mesenchymal stem cells combined with bone morphogenetic protein 2 effectively mitigates ovariectomy induced osteoporosis in rats
    Yang Jiujie, Zhao Wei, Wang Nan, Chen Xiaochun, Li Zhi, Ma Ji, Shen Dewei, Wang Yong, Niu Qingfei, Wang Tao, Zhou Yubo, Zhang Yang
    2020, 24 (1):  7-13.  doi: 10.3969/j.issn.2095-4344.1874
    Abstract ( 451 )   PDF (1051KB) ( 137 )   Save

    BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) secrete various cytokines and growth factors required for bone remodeling, which are considered to be excellent candidate cells for bone regeneration. Bone morphogenetic protein 2 (BMP2) and ADSCs have a synergistic effect on bone regeneration and can significantly enhance the osteogenic differentiation of ADSCs.

    OBJECTIVE: To explore the efficacy of conditioned medium of ADSCs combined with BMP2 on postmenopausal osteoporosis in a rat model.

    METHODS: Healthy and female Sprague-Dawley rats aged 8-10 months (n=75) were obtained from the Animal Center of Shenyang Medical College. Ovariectomy was performed to induce postmenopausal osteoporosis in 60 rats, and the remaining 15 rats underwent surgeries without removal of the ovaries (sham group). Ovariectomized rats were randomized into four groups: osteoporosis group, conditioned medium group, BMP2 group, and conditioned medium+BMP2 group, followed by injection of DMEM medium, ADSCs conditioned medium, BMP2, and ADSCs conditioned medium+BMP2 via the tail vein, respectively. After 12 weeks of treatment, femurs and serum samples of each group were taken. The number and structure of trabecular bone and trabecular spacing were detected histologically. Serum P1NP, ALP, TRAP, OPG, RANKL levels were detected by ELISA. Western blot and real-time PCR were used to detect RANKL and OPG at protein and mRNA levels. Cytokine chip analysis of cytokines in ADSCs conditioned medium was performed.

    RESULTS AND CONCLUSION: Compared with the sham group, increased trabecular spacing, fewer trabeculae and marked trabecular disconnection were observed in osteoporosis rats. The trabeculae in conditioned medium+BMP2 group appeared to be more complete and continuous with less widened spacing compared with the osteoporosis, conditioned medium and BMP2 groups. Serum levels of P1NP and ALP were dramatically higher, while TRAP level group was decreased significantly in the conditioned medium+BMP2 group compared with the other groups (both P < 0.05). In the conditioned medium+BMP2 group, RANKL/OPG ratio was reduced significantly compared with the other groups (P < 0.01), further promoting bone formation. ADSCs conditioned medium contained a variety of cytokines that were essential for bone formation and remodeling, including bone morphogenetic proteins 4 and 7, leukemia inhibitory factor, brain-derived neurotrophic factor, osteoprotegerin, and insulin-like growth factor 1. These results demonstrate that ADSCs conditioned medium combined with BMP2 can mitigate osteoporosis induced by ovariectomy and it may be an attractive strategy to treat postmenopausal osteoporosis.

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    Ammonium chloride lysis method versus non-lysis method for isolation of human adipose-derived stem cells
    Li Zifei, Wang Qian, Luan Jie, Mu Dali, Liu Chunjun, Xin Minqiang, Fu Su, Xu Boyang, Liu Wenyue, Chen Lin, Cheng Hao, Wang Chenglong, Kang Deni, Li Shangshan, Qi Jun
    2020, 24 (1):  45-50.  doi: 10.3969/j.issn.2095-4344.1869
    Abstract ( 559 )   PDF (797KB) ( 105 )   Save

    BACKGROUND: A general standard has not been established for the extraction and purification of adipose-derived stem cells (ADSCs). An erythrocyte lysis step for stromal vascular fraction is the commonly used method in the procedure for ADSCs isolation. However, there is a lack of evidence on whether this step will have adverse effects on human ADSCs (hADSCs).

    OBJECTIVE: To test the efficiency of two hADSCs isolation methods, which are erythrocyte-lysis method based on ammonium chloride and non-lysis method. Moreover, the biological characteristics of the hADSCs isolated by the two methods were also compared.

    METHODS: After collagenase digestion of lipoaspirate, erythrocyte lysis buffer was used to purify stromal vascular fraction in erythrocyte-lysis method, while in non-lysis method the buffer was not used. A MuseTM cell analyzer was used to assess living cell counting and proportion of stromal vascular fraction in both methods. Then hADSCs were cultured to the second passage for next testing. Cell morphology was observed under light microscope. Cell phenotype was detected by flow cytometry. Cell counting kit-8 was used to evaluate cell proliferation. Oil red O staining and alizarin staining were used to evaluate adipogenic and osteogenic ability of hADSCs after adipogenic and osteogenic induction. This study was approved by the Ethics Committee of the Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, and informed consents were signed by all participants.

    RESULTS AND CONCLUSION: (1) Compared with the erythrocyte lysis group, hADSCs obtained in the non-lysis group contained a larger number and a larger percentage of non-erythrocyte living cells. (2) The two groups of hADSCs were spindle-shaped and arranged as a fish shape. (3) The cell phenotypes of both groups met the phenotypic requirements for human mesenchymal stem cells. (4) The cell proliferation in the non-lysis group was faster than that in the erythrocyte lysis group, while there was no difference in the adipogenic and osteogenic abilities between the two groups. In conclusion, the use of erythrocyte lysis buffer reduces the isolation efficiency of hADSCs and inhibits cell proliferation. The non-lysis isolation method does not affect phenotypes, adipogenic and osteogenic ability of hADSCs. Therefore, it is not recommended to implement erythrocyte lysis during the isolation of hADSCs.

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    Allogeneic umbilical cord mesenchymal stem cell transplantation for metabolic syndrome in a tree shrew model
    Ruan Guangping, Yao Xiang, Cai Xuemin, Li Zian, Pang Rongqing, Pan Xinghua
    2020, 24 (1):  51-58.  doi: 10.3969/j.issn.2095-4344.1861
    Abstract ( 464 )   PDF (824KB) ( 175 )   Save

    BACKGROUND: Current treatments for metabolic syndrome are comprehensive treatments with drugs, aiming to improve patient’s life. Patients are required to have a high compliance to follow-up and have developed various adverse reactions. Therefore, there is no treatment to fundamentally delay the development of metabolic syndrome.

    OBJECTIVE: To establish a tree shrew model of metabolic syndrome and evaluation techniques, and to explore the therapeutic effect of umbilical cord mesenchymal stem cells in the tree shrew model, providing theoretical basis and reference method for clinical application of stem cell transplantation in metabolic syndromes.

    METHODS: Umbilical cord mesenchymal stem cells of tree shrews were obtained by adherent culture method, and met the biological characteristics of umbilical cord mesenchymal stem cells. The dark red fluorescent iodide DIR was used to label tree umbilical cord mesenchymal stem cells. Thirty-two tree shrews were fed high-sugar, high-cholesterol, high-salt diet and syrup diet, and injected streptozotocin to make metabolic syndrome models. Animal models were randomly divided into model control group (n=10) and cell treatment group (n=22). In the cell treatment group, the umbilical cord mesenchymal stem cells labeled in vitro were injected into the tail vein, and the model group was injected an equal volume of physiological saline in the meanwhile. Blood biochemical indicators, glucose tolerance, insulin resistance index and arterial blood pressure were measured after transplantation.

    RESULTS AND CONCLUSION: The tree shrew model of metabolic syndrome was successfully constructed, showing obvious insulin resistance, hyperglycemia, lipid metabolism disorder, and hypertension, which met the diagnostic criteria of metabolic syndrome. Umbilical cord mesenchymal stem cells transplantation could significantly reduce the blood glucose and blood lipid levels, improve insulin resistance and regulate insulin secretion in the tree shrew model of metabolic syndrome. Transplanted umbilical cord mesenchymal stem cells could be homing to the liver, kidney and pancreas of the tree shrew model of metabolic syndrome, and produce certain repairing effects.

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    Comparison of two passage methods affecting the transfection efficiency of human embryonic stem cells
    Sun Li, Zong Yanyan, Wei Jianfeng
    2020, 24 (1):  72-76.  doi: 10.3969/j.issn.2095-4344.1870
    Abstract ( 215 )   PDF (771KB) ( 68 )   Save

    BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency.

    OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection.

    METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively.

    RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.
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    The C-terminus of the amelogenin peptide promotes the proliferation of ALC ameloblasts through accelerating cell cycle
    Liu Min, Wang Ruijie, Song Danyang, Yang Sui, Tan Tao, Wang Lei, Wang Yixiang
    2020, 24 (1):  99-105.  doi: 10.3969/j.issn.2095-4344.1858
    Abstract ( 462 )   PDF (1164KB) ( 155 )   Save

    BACKGROUND: C-terminus of the amelogenin peptide (AMG-CP) is a small molecular endogenous peptide that is highly conserved among species. It is involved in important physiological processes during tooth development. Some studies have shown that AMG-CP can regulate the proliferation and differentiation of cementoblasts, bone marrow mesenchymal stem cells and periodontal ligament fibroblasts, but the biological function of AMG-CP on ameloblasts has not been elucidated.

    OBJECTIVE: To investigate the effects of different concentrations of AMG-CP on the proliferation of ALC ameloblasts and its underlying mechanisms.

    METHODS: AMG-CP was successfully synthesized and determinated by liquid chromatography and mass spectrometry. The effects of AMG-CP at 0, 0.5, 1, 2 mg/L on the proliferation of ALC ameloblasts were observed by xCELLigence RTCA cell analysis system in real time. The effect of AMG-CP at 0, 1, 2 mg/L on cell cycle of ALC was detected by flow cytometry. Real-time PCR was used to detect the expression of cyclin D1, CDK4, MCM2, MCM5 mRNA in ALC cells treated with AMG-CP at 0, 1, 2 mg/L. Western blot was carried out to evaluate the effect of AGM-CP at 0, 1 mg/L on MAPK-ERK1/2 pathway by detecting the expression of phosphorylated ERK1/2 and total ERK1/2 in ALC cells. Pathway blockade assay was performed by using ERK1/2 blocker U0126 to pretreat ALC cells. Then cell proliferation ability as well as phosphorylated ERK1/2 expression was analyzed by xCELLigence RTCA cell analysis system and western blot.

    RESULTS AND CONCLUSION: Compared with the control group, AMG-CP promoted the proliferation of ALC cells, and decreased the population doubling time in a dose-depending manner. Flow cytometry detected the acceleration of cell cycle after treatment with AMG-CP. The results of Real-time PCR showed that AMG-CP upregulated cell cycle-related genes (cyclin D1, CDK4, MCM2, MCM5) expression. Western blot results showed that AMG-CP could upregulate the expression of phosphorylated ERK1/2 and activate MAPK-ERK1/2 signaling pathway in ALC cells. After U0126 was used to inhibit the MAPK-ERK1/2 pathway, the ability of AMG-CP promoting ALC proliferation was inhibited. These results suggest that AMG-CP has a potential to activate MAPK-ERK1/2 pathway, accelerate the process of cell cycle, and then promote the proliferation of ALC cells, all of which indicate that AMG-CP has the potential to promote the proliferation of ameloblasts.

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    Effect of microRNA-132-3p overexpression on proliferation and osteogenic differentiation of MC3T3-E1 cells
    Sun Fen, Liu Mingyan, Liu Ming, Sun Xinyi, Feng Yunxia
    2020, 24 (1):  59-64.  doi: 10.3969/j.issn.2095-4344.1844
    Abstract ( 561 )   PDF (796KB) ( 88 )   Save

    BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p.

    OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation.

    METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay.

    RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.

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    Analysis of full-length sequence and 18 point mutations of HLA-B in a leukemia patient and her family
    Zhong Yanping, Zou Hongyan, Quan Zhanrou, Deng Zhihui, Hong Wenxu
    2020, 24 (1):  77-82.  doi: 10.3969/j.issn.2095-4344.1859
    Abstract ( 545 )   PDF (986KB) ( 160 )   Save

    BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly.

    OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology.

    METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes.

    RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3'UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.

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    In vitro differentiation potential of human urine-derived stem cells into neuron-like cells and their protective effects against spinal cord injury in rats
    Deng Ming, Xie Ping, Wu Fei, Ma Yonggang, Zhou Yan, Chen Qing, Liu Shiqing, Ming Jianghua
    2020, 24 (1):  93-98.  doi: 10.3969/j.issn.2095-4344.2005
    Abstract ( 555 )   PDF (790KB) ( 64 )   Save

    BACKGROUND: Human urine-derived stem cells are newly discovered adult stem cells, characterized by rich sources, simple extraction, good proliferative ability and multi-directional differentiation potential. In recent years, human urine-derived stem cells have been used for the repair of neurological functions in urinary diseases, such as stress urinary incontinence and vesicoureteral reflux. 

    OBJECTIVE: To explore the biological characteristics of human urine-derived stem cells and to study their repairing effect in a rat model of spinal cord injury.

    METHODS: Cell phenotypes of human urine-derived stem cells were detected using flow cytometry, and the immunohistochemical staining was used to identify neuron-like cells differentiated from human urine-derived mesenchymal stem cells. Then, an animal model of spinal cord injury at T9 segment was made by Allen method, and after modeling 24 Sprague-Dawley rats were assigned into spinal cord injury group or cell treatment group (n=12/group). In the cell treatment group, the model rats were injected 2 μL of 1.0×1011/L human urine-derived stem cells, while in the spinal cord injury group, the rats were administered the same volume of L-DMEM containing 10% fetal bovine serum. Basso, Beattie and Bresnahan scores were valued at 1, 10, 20, and 30 days after modeling. Spinal cord samples from all the rats were taken out at 30 days after modeling, and Luxol Fast Blue staining, microglia/macrophages staining and glial fibrillary acidic protein staining were used to value the injured area of the spinal cord and the fluorescence intensity of glial fibrillary acidic protein.

    RESULTS AND CONCLUSION: (1) Flow cytometry showed high expression on CD29 and CD90, and low expression on CD45 in human urine-derived mesenchymal stem cells. Moreover, human urine-derived mesenchymal stem cells could be induced to differentiating into neuron-like cells in vitro. (2) Basso, Beattie and Bresnahan scores showed no significant difference between the two groups at 1 and 10 days after modeling (P > 0.05), while, at 20 and 30 days after modeling, the scores in the cell treatment group were significantly higher than those in the spinal cord injury group (P < 0.05). (3) Luxol Fast Blue staining showed that the injured area of the spinal cord in the cell treatment group was markedly less than that in the spinal cord injury group (P < 0.05), and the glial fibrillary acidic protein showed lower fluorescence intensity in the cell treatment group than the spinal cord injury group (P < 0.05). To conclude, human urine-derived stem cells can differentiate into neuron-like cells and have therapeutic effects in the rat model of spinal cord injury. 

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    Changes of peripheral and bone marrow erythrocytes in plateau pikas and rats after hypoxia exposure
    Ma Jie, Ji Linhua, Li Zhanquan, Liu Huihui, Ma Lan, Xi Rui, Cui Sen
    2020, 24 (1):  112-117.  doi: 10.3969/j.issn.2095-4344.2008
    Abstract ( 482 )   PDF (909KB) ( 59 )   Save

    BACKGROUND: Changes of red blood cell in peripheral blood and bone marrow erythropoietic system in plateau pikas are of great significance for hypoxic adaptation.

    OBJECTIVE: To explore the hypoxic adaptation of erythropoietic system in plateau pikas by comparing the morphological changes of peripheral blood and bone marrow between plateau pikas and rats exposed to hypoxia.

    METHODS: There were 12 healthy wild plateau pikas and 12 clean Sprague-Dawley rats and were randomly divided into experimental and control groups, 6 in each group. The experimental group animals were fed in a simulated 5 000 m altitude hypobaric hypoxia chamber for 28 consecutive days, and the control group animals were fed in the laboratory at 2 260 m altitude.

    RESULTS AND CONCLUSION: (1) The diameter of the erythrocytes was smaller and red blood cell count was higher in plateau pikas than those in the rats of control group. After 28 days of hypoxia, red blood cell count, hemoglobin and hematocrit were increased in both experimental groups (P < 0.001), but the increased rate of plateau pikas were less than that of the rats. The mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration showed no significant changes in plateau pikas. (2) Results of bone marrow smear showed that the proportion of polychromatic and orthochromatic erythroblasts had no significant changes in plateau pikas after hypoxia, but increased significantly in the rats (P < 0.05). (3) Hematoxylin-eosin staining results of the sternum indicated that the immature erythroblasts islands did not change significantly in plateau pikas, but increased significantly in rats. (4) So the erythroid changes in peripheral blood and bone marrow of plateau pikas before and after hypoxia are significantly lower than those of the Sprague-Dawley rats, and they may be related to the hypoxia adaptation mechanism.

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    A metrics method for cellular network structure
    Zhang Yu, Liu Fang
    2020, 24 (1):  83-86.  doi: 10.3969/j.issn.2095-4344.1865
    Abstract ( 383 )   PDF (521KB) ( 91 )   Save

    BACKGROUND: Communication between cells is indispensable in a multicellular organism society. Many cells communicate with each other, forming a complex cellular network structure. It is very important to measure and evaluate the relevant attribute values of cellular network structure.

    OBJECTIVE: To propose a metrics method of cellular network structure based on a complex network.

    METHODS: Based on literature research and practical application, the metrics framework of cellular network structure was established. The structure of cellular network was measured in the aspects of the degree of node, the degree distribution of cellular network, the average path length of cellular network and the cluster coefficient of cellular network. A small experiment was taken as an example to verify the validity of the method.

    RESULTS AND CONCLUSION: In the degree distribution of cellular networks, the degree values of most cell nodes are relatively small, and only a small number of cell nodes have higher degree values. The more obvious power-law distribution of the degree distribution P(k) of cell nodes indicates the more reasonable structure of the cellular network as well as the more normal cellular network. At the same time, many cellular network structures have smaller average path lengths. The larger cluster coefficient of the cellular network indicates the higher aggregation characteristics of the cellular network. Generally speaking, the tighter the cell network structure is, the more obvious the clustering characteristics of the cell network structure are, and the more normal the cellular network is.
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    Spinal cord homogenate promotes neurite outgrowth through activation of formyl peptide receptor 2 after spinal cord injury
    Zhang Liang, Liu Mingyong, Liu Peng, Xue Xin, Chen Zongfeng, Zhang Liangmin, Zhang Jian, Guo Qiaonan, Zhao Jianhua
    2020, 24 (1):  106-111.  doi: 10.3969/j.issn.2095-4344.1868
    Abstract ( 494 )   PDF (793KB) ( 178 )   Save

    BACKGROUND: Previous studies have observed the expression of formyl peptide receptor 2 in newly differentiated neurons from neural stem cells and confirmed that formyl peptide receptor 2 can promote the migration of neural stem/progenitor cells and induce them to differentiate into neurons. Formyl peptide receptor 2 ligands are present in damaged spinal cord tissues, but the binding of different ligands with FPR2 may lead to different and even opposite biological effects.

    OBJECTIVE: To investigate the neurite outgrowth after the binding of the ligands produced following spinal cord injury with formyl peptide receptor 2.

    METHODS: The fetal rat cerebral cortical neurons were extracted by enzymatic digestion. Spinal cord injury models were established in Sprague-Dawley rats, and the injured spinal cord homogenate was extracted. (1) Experiment 1: To observe the effect of the activation of formyl peptide receptor 2 on neurite outgrowth, the cells were divided into control group, formyl peptide receptor 2 blocker group (addition of WRW4), spinal cord homogenate group, spinal cord homogenate+WRW4 group. (2) Experiment 2: To observe the effect of blockade of AKT and ERK signaling pathways on neurite outgrowth after activation of formyl peptide receptor 2, the cells were divided into control group, AKT and ERK signaling pathway blocker group (addition of Ly294002+PD98059), spinal cord homogenate group, spinal cord homogenate+ Ly294002+PD98059 group. After 24 hours of culture, adherent neurons were treated with above-mentioned regimens for 7 days. Immunofluorescence staining with confocal microscope detection was used to observe the effect of spinal cord homogenate on neurite outgrowth via the activation of formyl peptide receptor 2. The cells were treated by the above-mentioned regimens for 30 minutes and phosphorylated protein levels were detected by western blot. The cells were treated with the above-mentioned regimens for 24 hours, and western blot assay was used to detect F-actin levels and observe the phosphorylation of key proteins in MAPK and PI3K/Akt pathways with the presence of formyl peptide receptor 2 specific blocker WRW4.

    RESULTS AND CONCLUSION: WRW4 could eliminate the effects of injured spinal cord homogenates on neurite outgrowth, including neurite length, the number of primary neurites, and the number of branch points. Spinal cord homogenate increased the phosphorylation of ERK1/2 and Akt in neurons, whereas this effect could be blocked by WRW4. Ly294002 and PD98059 could also eliminate the effects of homogenates on the neurite outgrowth. Spinal cord homogenate significantly increased the expression of F-actin in neurons, but this effect was blocked by WRW4. These results suggest that spinal cord homogenates can expedite neurite outgrowth by activating formyl peptide receptor 2, which may be related to the increased phosphorylation of ERK1/2 and Akt.

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    Effects of cytokine-induced neutrophil chemoattractant 3 on the survival and proliferation of neural stem cells
    Sun Nai, Wang Dali, Zhang Jun, Cao Jinming, Qiu Fucheng, Liu Huimiao, Li Dong, Gu Ping
    2020, 24 (1):  118-123.  doi: 10.3969/j.issn.2095-4344.2004
    Abstract ( 446 )   PDF (696KB) ( 56 )   Save

    BACKGROUND: Our previous study was the first to find that bone marrow stromal cells could secrete neutrophil chemokine-3. Moreover, the expression of cytokine-induced neutrophil chemoattractant 3 (CINC-3) could be up-regulated by 1.5 times during the differentiation of neural stem cells into neurons regulated by bone marrow stromal cells. These results suggest that CINC-3 may promote the proliferation and differentiation of neural stem cells.

    OBJECTIVE: To observe the effect of CINC-3 on survival and proliferation of hippocampal neural stem cells in neonatal rats.

    METHODS: Hippocampal neural stem cells from neonatal Sprague-Dawley rats were isolated and cultured in vitro, and passage 3 neural stem cells were divided into control and CINC-3 groups. The survival rate of the cells was measured by MTS method, cell survival and proliferation were observed using cell growth curve and live/dead cell staining, and the expression of Nestin in neural stem cells was detected by real-time PCR and immunofluorescence staining for observation of neural stem cell proliferation.

    RESULTS AND CONCLUSION: (1) When the concentration of CINC-3 increased from 1 to 20 μg/L, the survival rate of neural stem cells increased gradually. When the concentration of CINC-3 was 10 μg/L, the survival rate of neural stem cells was the highest and the cell viability was the best (P < 0.05). When the concentration of CINC-3 further increased to 20 μg/L, there was no significant difference in the survival rate of neural stem cells. (2) The positive rate of Nestin in the CINC-3 group was significantly higher than that in the control group (P < 0.05). (3) The expression of Nestin mRNA in the CINC-3 group was significantly higher than that in the control group (P < 0.05). These findings indicate CINC-3 can promote the survival and proliferation of hippocampal neural stem cells.

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    Mechanical properties and cytoskeletal protein changes after ANLN deficiency
    Xu Limeng, Luo Wenyu, Zhang Shuwei, Wang Li
    2020, 24 (1):  87-92.  doi: 10.3969/j.issn.2095-4344.1875
    Abstract ( 462 )   PDF (769KB) ( 57 )   Save

    BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported.

    OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells.

    METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy.

    RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.

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    Disulfiram combined with cooper inhibits proliferation and induces apoptosis of osteosarcoma
    Xu Chaojian, Zhang Long, Cheng Caitong, Sun Xiaojuan, Lü Zhi
    2020, 24 (1):  124-129.  doi: 10.3969/j.issn.2095-4344.1853
    Abstract ( 517 )   PDF (980KB) ( 71 )   Save

    BACKGROUND: Studies have shown that disulfiram has anti-tumor activity, which can be combined with copper (Cu) ions to exert an anti-tumor effect on multiple tumors in vivo and in vitro, but the effect of disulfiram on osteosarcoma proliferation and apoptosis has not been clarified.

    OBJECTIVE: To investigate the effect of disulfiram combined with Cu on osteosarcoma proliferation and apoptosis and the possible mechanism in vivo and in vitro.

    METHODS: The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University (approval No. 2017LL077). (1) In vitro study: diethyldithiocarbamate (DDTC)-Cu (0.5, 1, 2, 3, and 5 μmol/L) was configured with Cu and DDTC which is the transformation of disulfiram after absorbed by bodies. DDTC single drug (5 μmol/L), Cu single drug (5 μmol/L) and blank control groups were set. Osteosarcoma cell lines Saos-2 and MG-63 were treated with drugs, and cell counting kit-8 assay was used to detect the inhibitory effect of DDTC-Cu at different concentrations on the proliferation of Saos-2 and MG-63 cells. Changes in Saos-2 apoptosis were measured by AnnexinV-FITC/PI staining. (2) In vivo study: A total of 10 BALB/c-nu/nu female nude mice of 4 weeks old were randomly divided into DDTC-Cu group and control group. The mixture of Saos-2 cells and Matrigel (1:1 mixed, 400 μL per mouse) was injected subcutaneously into the right back of nude mice. Two weeks after inoculation, model mice were intraperitoneally injected dexamethasone (0.5 mg/kg once every other day) in the control group, and dexamethasone (0.5 mg/kg once every other day) and DDTC-Cu complex (10 nmol/g once every other day) in the DDTC-Cu group. Xenograft tumors in each group were measured at regular intervals and tumor growth curves were drawn. Five weeks after inoculation, the animals were sacrificed under anesthesia, and tumors were completely removed. Immunohistochemistry was used to detect the expression level of ki67 protein in tumor paraffin sections. The expressions of proteins related to cell proliferation and apoptosis and JNK pathway proteins were determined by western blot analysis.

    RESULTS AND CONCLUSIONS: (1) In vitro study: The proliferation inhibition in the DDTC-Cu group was significantly stronger than that in the DDTC single drug, Cu single drug and blank control groups. Cell counting kit-8 results showed that DDTC-Cu inhibited osteosarcoma proliferation in a dose-dependent manner, with 50% inhibiting concentration of 0.337 μmol/L (Saos-2) and 0.487 μmol/L (MG-63) for 24 hours, respectively. The results of flow cytometry showed that DDTC-Cu promoted Saos-2 apoptosis in a dose-dependent manner. (2) In vivo study: The tumor volume and mass of the DDTC-Cu group were smaller than those of the control group. Immunohistochemical results showed that the expression level of ki-67 protein in the DDTC-Cu group was lower than that in the control group. Western blot results showed that the expression levels of p-JNK and c-jun in the DDTC-Cu group were up-regulated. To conclude, disulfiram combined with Cu inhibits proliferation and induces apoptosis of osteosarcoma in vitro and in vivo, and its mechanism may be related with activation of JNK signaling pathway.

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    Rhodiola intervention improves mitochondrial autophagy and fusion-division in skeletal muscle cells of mice with high intensity exercise
    Cao Haixin, Wang Xiaomei
    2020, 24 (1):  136-140.  doi: 10.3969/j.issn.2095-4344.1852
    Abstract ( 447 )   PDF (658KB) ( 65 )   Save

    BACKGROUND: Excessive exercises cause a large accumulation of oxidative active substances in the body to damage skeletal muscle cells. Mitochondria play a key role in energy metabolism during exercise. Studies have shown that Rhodiola can reduce the level of lipid peroxidation in muscle tissue and protect damaged endothelial cells.

    OBJECTIVE: To explore the mechanism underlying Rhodiola improving skeletal muscle function of mice with high intensity exercise by regulating mitochondrial function.

    METHODS: The study protocol was approved by the Ethics Committee of Xi’an Shiyou University in China. Forty BALB/c mice were divided into blank control group, exercise group, Rhodiola control group and Rhodiola intervention group. Mice in the blank control had no exercise and intervention. Mice in exercise group were given intragastric administration of normal saline followed by high intensity exercise. Mice in Rhodiola intervention group and Rhodiola control group were given intragastric administration of the mixture of Rhodiola and normal saline, followed by exercise or not. The interventions were performed once a day for 28 consecutive days. Body mass, forearm grip strength and exhaustion time were observed. Western blot assay was used to detect expression of manganese superoxide dismutase protein, p53 protein, mitochondrial origin and autophagy-associated protein in the skeletal muscle. RT-qPCR was used to detect skeletal muscle Mfn-1, Mfn-2, Opa-1, Drp-1, and fis-1 mRNA expression.  

    RESULTS AND CONCLUSION: (1) From the 2nd week, the grip strength of forelimbs in the exercise group was significantly lower than that in the other three groups (P < 0.05), but there was no significant difference among blank control group, Rhodiola control group, and Rhodiola intervention group (P > 0.05). (2) At the 3rd and 4th weeks, the exhaustion time of weight-bearing swimming training was significantly shorter in the exercise group than the Rhodiola intervention group (P < 0.05). (3) The levels of manganese superoxide dismutase protein and p53 protein in skeletal muscle cells of mice in the exercise group were significantly higher than those in the other groups (< 0.05). The levels of manganese superoxide dismutase protein and p53 protein in skeletal muscle cells of mice in the Rhodiola intervention group were significantly higher than those in the Rhodiola control group (P < 0.05). (4) Compared with the blank control group, the levels of PGC-1a and LC3-II/LC3-I in skeletal muscle cells of mice in the exercise group increased significantly, while the levels of Atg7 and P62 decreased significantly (P < 0.05). Compared with the Rhodiola control group, the levels of PGC-1a and LC3-II/LC3-I in skeletal muscle cells of mice in the Rhodiola intervention group increased significantly, while the levels of Atg7 and P62 decreased significantly (P < 0.05). (5) Compared with the blank control group, the expression of fusion gene and Drp-1 gene in the exercise group decreased and increased, respectively, but there was no significant difference between the two groups (P > 0.05). Compared with the blank control group, the Rhodiola exercise intervention group also showed a downward trend in the expression of fusion gene and an upward trend in the expression of Drp-1 mRNA, but there was no significant difference between the two groups (P > 0.05). To conclude, Rhodiola can significantly improve the exercise endurance of mice with high intensity exercise, which may be related to the improvement of skeletal muscle mitochondrial autophagy, origin and fusion-division.

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    Effect of sex combing protein 1 on proliferation and differentiation of osteoblasts in inflammatory microenvironment#br#
    Xu Huijun, Shi Dongmei, Zhang Mi, Wu Saixuan, Dong Ming, Lu Ying, Niu Weidong
    2020, 24 (1):  130-135.  doi: 10.3969/j.issn.2095-4344.1845
    Abstract ( 367 )   PDF (690KB) ( 49 )   Save

    BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported.

    OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment.

    METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA.

    RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction.

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    Factors affecting the secretion of vascular endothelial growth factor from dental pulp stem cells
    He Hongzhi, Ma Dandan
    2020, 24 (1):  141-147.  doi: 10.3969/j.issn.2095-4344.1860
    Abstract ( 398 )   PDF (741KB) ( 91 )   Save

    BACKGROUND: How to regulate the secretion of vascular endothelial growth factor from dental pulp stem cells is of great significance for promoting dental pulp regeneration by dental pulp stem cells, especially promoting dentinogenesis, dental pulp angiogenesis and neurogenesis.

    OBJECTIVE: To review the factors affecting the secretion of vascular endothelial growth factor from dental pulp stem cells, providing ideas for pulp regeneration and other clinical applications.

    METHODS: We searched the articles in PubMed, CNKI, WanFang databases with the keywords of “vascular endothelial growth factor; dental pulp stem cell; dental pulp regeneration; hypoxia; inflammatory mediator; bacterial virulence factor; growth factor; material” in Chinese and English, respectively. Finally, 56 articles met the criteria for review.

    RESULTS AND CONCLUSION: Vascular endothelial growth factor is the most important cytokine in angiogenesis and neovasculization, which promotes the proliferation and differentiation of stem cells as well as protecting nerves and promoting neurogenesis. Dental pulp stem cells are the most important stem cells in dental pulp tissues. They are also important seed cells in pulp regeneration. Dental pulp stem cells have biological characteristics such as high proliferation, self-renewal and multi-lineage differentiation, and have certain secretory activities, which can be used as an alternative source of exogenous vascular endothelial growth factor. A variety of factors, such as hypoxia, bacterial virulence factors, inflammatory factors, growth factors and materials, are associated with the cytokine secretion activity of dental pulp stem cells, which can affect the expression and secretion of vascular endothelial growth factor in dental pulp stem cells. Therefore, increasing concern has been emphasized on the regulation of vascular endothelial growth factor ecpression and secretion in dental pulp stem cells and the better use in pulp regeneration.


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    Research progress of mesenchymal stem cells in treating lung injury
    Zhang Yunqiang, Ke Xixian, Zhang Lingtao, Liang Guiyou
    2020, 24 (1):  148-153.  doi: 10.3969/j.issn.2095-4344.1855
    Abstract ( 604 )   PDF (693KB) ( 287 )   Save

    BACKGROUND: Mesenchymal stem cell therapy, as a new treatment strategy, has great treatment potential for lung injury.

    OBJECTIVE: To review the roles and protective mechanisms of mesenchymal stem cells in the treatment of lung injury, providing theoretical basis for clinical treatment of lung injury with mesenchymal stem cells.

    METHODS: We searched the articles about the treatment of mesenchymal stem cells for lung injury from May 2001 to May 2019 in WanFang, CNKI, PubMed and Web of Science databases. The retrieval terms were “mesenchymal stem cells, lung injury, pulmonary injury, lung” in Chinese and English. After excluding old and repetitive articles, a total of 53 articles were included for further analysis.

    RESULTS AND CONCLUSION: (1) After summarizing the definition and characteristics of mesenchymal stem cells and its mechanism of treating lung injury, we found that mesenchymal stem cells can treat lung injury by their own functions and by producing various cytokines and exosomes. (2) The related signaling pathways of mesenchymal stem cells in the treatment of lung injury are summarized, such as: PI3K/AKT signaling pathway, Wnt signaling pathway and nuclear factor-κB signaling pathway. (3) Combined use of mesenchymal stem cells and other drugs, such as erythropoietin and corticosteroids, can enhance the therapeutic effects on lung injury. (4) This article can provide theoretical basis for the treatment of lung injury with mesenchymal stem cells in the clinical practice.
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    New insight into cell transplantation for repairing intervertebral discs
    Liu Tingting, Han Changxu, Wang Guoqiang
    2020, 24 (1):  154-158.  doi: 10.3969/j.issn.2095-4344.2003
    Abstract ( 499 )   PDF (641KB) ( 109 )   Save

    BACKGROUND: Current treatment of disc degeneration is limited to palliative care or active surgical intervention, but neither can it slow or reverse disease progression, nor is the long-term efficacy satisfactory. In recent years, with the continuous development of tissue engineering and regenerative medicine, the use of cell therapy to repair degenerative intervertebral discs has attracted more and more researchers’ attention.

    OBJECTIVE: To summarize the current status of cell transplantation in the treatment of intervertebral disc degeneration, and to provide theoretical basis and experimental basis for future research.

    METHODS: The relevant articles were retrieved from the CNKI, WanFang and PubMed databases by computer. The search time was set from January 2001 to January 2019. The keywords were “intervertebral disc degeneration, cell therapy, stem cells, cell transplantation” in Chinese and English, respectively. Articles related to stem cell treatment of intervertebral disc degeneration, especially focusing on the latest experimental and clinical research results, were included.

    RESULTS AND CONCLUSION: Degeneration of the intervertebral disc is mainly caused by the decreased vitality and quantity of nucleus pulposus cells, the reduction of proteoglycan synthesis, the dehydration of nucleus pulposus and the increase of metabolic waste. Continuous explorations on the therapeutic mechanism by which cell transplantation promotes nucleus pulposus regeneration cannot only provide a more detailed understanding of the pathogenesis and repair mechanism of intervertebral disc degeneration, but also give new strategies for the prevention and cell therapy of other related diseases.
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