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28 July 2019, Volume 23 Issue 21 Previous Issue    Next Issue

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Roles of ROCK signaling in proliferation and paracrine action of hypoxia-induced c-Kit+ bone marrow mesenchymal stem cells Shao Zhongming, Wang Keke, Liao Xiaomin, Ha Yanping, Li Rujia, Shen Zhihua, Jie Wei 2019, 23 (21):  3281-3288.  doi: 10.3969/j.issn.2095-4344.1742 Abstract ( 233 )   PDF (952KB) ( 307 )   Save BACKGROUND: Hypoxia stimulates the proliferation of bone marrow mesenchymal stem cells, but the specific mechanism is still unclear. The Rho-associated coiled-coil containing kinases (ROCK) signaling has an important influence on cell proliferation, migration and apoptosis. Few studies have been reported on the effect of ROCK signaling on the biological functions of bone marrow mesenchymal stem cells. OBJECTIVE: To analyze the effect of ROCK signaling on the hypoxia-induced proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells. METHODS: Rat bone marrow mesenchymal stem cells derived from the rat femur were obtained by adherent method, and the c-Kit+ subpopulation was sorted by magnetic activated cell sorting. Cells were cultured under normoxia (21% O2) and hypoxia (2% O2). After treatment with an ROCK signal inhibitor Fasudil (10 μmol/L), western blot was used to detect the expression of p-MLC (T18/S19), MLC, p-MLC2(T18/S12)/MLC2, PCNA and cell cycle related proteins. The cell proliferation was analyzed by cell counting, the cell cycle was detected using flow cytometry, and the changes of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor in the cell supernatant were detected by ELISA.  RESULTS AND CONCLUSION: c-Kit+ subpopulation were successfully isolated from rat bone marrow mesenchymal stem cells. Compared with normoxia, hypoxia treatment significantly down-regulated MLC expression, up-regulated p-MLC(T18/S19) expression and therefore increased the ratio of p-MLC(T18/S19)/MLC2. Meanwhile, hypoxia promoted the proliferation of c-Kit+ bone marrow mesenchymal stem cells, as evidenced by the increase in cell number and PCNA protein expression. Fasudil inhibited the hypoxia-induced the proliferation of c-Kit+ bone marrow mesenchymal stem cells, arrested the cell cycle at S phase, inhibited CDK2 and CDK4 and increased p16 expression. Hypoxia significantly promoted the secretion of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor by c-Kit+ bone marrow mesenchymal stem cells, and Fasudil attenuated hypoxia-induced vascular endothelial growth factor and transforming growth factor β1 expression but astonishingly upregulated basic fibroblast growth factor level. These results indicate that hypoxia promotes cell proliferation and paracrine of angiogenic factors and activates ROCK signaling in c-Kit+ bone marrow mesenchymal stem cells; Fasudil inhibits hypoxia-induced proliferation and expression of angiogenic factors in c-Kit+ bone marrow mesenchymal stem cells, suggesting that ROCK signaling plays a positive role in the proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells. Figures and Tables | References | Related Articles | Metrics

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Effects of red light-emitting diodes with different light energies on proliferation of human periodontal ligament stem cells and apical papillary stem cells Wu Yan, Yang Yaoyao, Wang Yao 2019, 23 (21):  3289-3293.  doi: 10.3969/j.issn.2095-4344.1744 Abstract ( 547 )   PDF (656KB) ( 111 )   Save BACKGROUND: Photobiomodulation is a new treatment option. Whether red light-emitting diodes can affect the proliferation and regeneration of dental stem cells is the key to exploring new treatments for maxillofacial regeneration. OBJECTIVE: To compare the effects of red light-emitting diodes with different light energies on the proliferation of human periodontal ligament stem cells and apical papillary stem cells. METHODS: Periodontal ligament stem cells and apical papillary stem cells were isolated and cultured by flow cytometry. Proliferation of these cells was detected using MTT assay after exposure to red light-emitting diodes at different light energies (0, 1, 3, and 5 J/cm2).  RESULTS AND CONCLUSION: Apical papillary stem cells proliferated faster than periodontal ligament stem cells under non-lighting environment. Red light emitting diodes at 1, 3, and 5 J/cm2 promoted the proliferation of two kinds of odontogenic stem cells. The red light-emitting diode promoted the proliferation of periodontal ligament stem cells at proliferative and logarithmic growth phase (1-7 days). And 5 J/cm2 red light emitting diode had the most obvious effect in early stage, and 1 J/cm2 had the strongest effect in the late stage. The red light-emitting diode had a significant effect on the logarithmic growth (5-7 days) of apical papilla stem cells, and under 3 J/cm2 red light-emitting diode the proliferation peaked on the 7th day. Therefore, the red light-emitting diode can promote the proliferation of human periodontal ligament stem cells and apical papillary stem cells in dose- and time-dependent manner. Exposure to red light-emitting diode at proper energy and time can promote cell proliferation quickly and effectively. Figures and Tables | References | Related Articles | Metrics

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Bone marrow mesenchymal stem cell-derived exosomes reduce the activation of type A1 astrocytes after spinal cord injury Zhou Yan, Wang Lin, Pei Shuang, Li Yanfei, Chen Xuemei, Jia Yanjie 2019, 23 (21):  3294-3301.  doi: 10.3969/j.issn.2095-4344.1760 Abstract ( 530 )   PDF (1130KB) ( 346 )   Save BACKGROUND: Previous studies found that exosomes from bone marrow mesenchymal stem cells could reduce the number of reactive astrocytes after spinal cord injury, thereby improving post-injury motor function. OBJECTIVE: To explore the role of bone marrow mesenchymal stem cell-derived exosomes in the activation of A1 astrocytes as well as in the repair of spinal cord injury.  METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawely rats were cultured, and then the exosomes from the cell supernatant were extracted and identified by ultracentrifugation. Eighty adult male Sprague-Dawley rats were randomly divided into sham operation group, spinal cord injury group (model group), bone marrow mesenchymal stem cell group (cell transplantation group) and exosome group (n=20/group). Except the sham operation group, an animal model of spinal cord injury was made in all the groups by Allen method. Two hours postoperatively, the equal volume of PBS, bone marrow mesenchymal stem cell suspension or bone marrow mesenchymal stem cell-derived exosome suspension was injected through the tail vein respectively, every 3 days, for three sessions. Basso, Beattie and Bresnahan scoring for motor function of the hind limb was performed preoperatively and and 1, 3, 7, 14, 28 days postoperatively. Hematoxylin-eosin staining and transmission electron microscope were used to observe the morphology of spinal cord tissue at 7 days postoperatively. Immunofluorescence staining was used to detect the number of A1 astrocytes, NeuN positive neurons and Caspase-3 positive apoptotic cells. ELISA assay was used to detect the levels of tumor necrosis factor-α and interleukin-1α in the spinal cord tissue. Western blot assay was used to determine relative protein expression levels of C3 and GFAP. RESULTS AND CONCLUSION: Exosomes from bone marrow mesenchymal stem cells showed a saucer-like appearance under transmission electron microscope, expressing exosome markers CD63 and CD9. The exosomes reduced inflammatory cell infiltration of spinal cord tissue, reduced myelin degeneration and axonal degeneration, reduced the expression levels of tumor necrosis factor-α, interleukin-1α and C3, reduced the number of A1 astrocytes, and reduced cell apoptosis, but increased the number of residual neurons, and improved limb motor function. To conclude, exosomes from bone marrow mesenchymal stem cells can reduce the activation of type A1 astrocytes in the spinal cord and promote the recovery of motor function after spinal cord injury. Figures and Tables | References | Related Articles | Metrics

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Liposome- versus lentivirus-mediated transfection of P75 nerve growth factor receptor and nerve growth factor into bone marrow mesenchymal stem cells Zhu Lunjing, Bei Chaoyong, Duan Jiangtao, Peng Chengfei, Ma Yuegang, Wang Ning, Chen Junyi 2019, 23 (21):  3302-3308.  doi: 10.3969/j.issn.2095-4344.1759 Abstract ( 338 )   PDF (852KB) ( 134 )   Save BACKGROUND: As a receptor for nerve growth factor (NGF), P75 neurotrophin receptor (P75NTR) can mediate distinct signaling pathways in different cells, but the regulatory effect in bone marrow mesenchymal stem cells has not been studied yet. Liposome and lentivirus-mediated single-gene transfection cell technology have matured, but the differences in mediating double gene transfection have not been reported. OBJECTIVE: To construct the overexpression plasmid and lentiviral vector of rat P75NTR and NGF, and to compare the effect and practicability of transfected bone marrow mesenchymal stem cells by lipofection and lentivirus infection in order to select the best experimental solution according to different experimental requirements in the future.  METHODS: The gene primers of rat P75NTR and NGF were designed. Genomic DNA was extracted for PCR amplification, and GV358-P75NTR and GV492-NGF overexpression plasmids were constructed by recombinant plasmid vector, transfected into 293T cells, and ultracentrifuged by lentiviral vector to collect target genes. The lentivirus was expressed and the titer of the virus sample was determined. The rat primary bone marrow mesenchymal stem cells were cultured in vitro, and one group was co-transfected with plasmid GV358-P75NTR and GV492-NGF with liposome Lipo3000, and the other group was treated with lentivirus. Negative control group and blank control group were set at the same time. The transfected bone marrow mesenchymal stem cells were routinely cultured. The expression of red and green fluorescence was observed under fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75NTR and NGF proteins was detected by western blot. Bone marrow mesenchymal stem cells were digested and subcultured on day 7 after transfection. The expression of red and green fluorescence was observed by fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75NTR and NGF proteins was detected by western blot.  RESULTS AND CONCLUSION: The GV358-P75NTR and GV492-NGF overexpression plasmids and lentivirus were constructed in accordance with the experimental design. For the abundance of red-green fluorescence in the microscope field, the liposome co-transfection group increased at first and then decreased; the lentivirus co-transfection group continued to increase; and the lentivirus co-transfection group was higher than the liposome co-transfection group at each time point. In the liposome co-transfection group, the fluorescence expression of the subcultured cells after transfection was lower than that of the non-subcultured cells, and the lentivirus co-transfection group showed no changes. The expression of P75NTR and NGF proteins in the lentiviral infection group was significantly higher than that in the liposome group at each time point. The expression levels of P75NTR and NGF proteins in the two target gene transfection groups were significantly higher than those in the negative control group and blank group. Both P75NTR and NGF genes could be transfected into rat bone marrow mesenchymal stem cells via liposome and lentivirus co-transfection. Compared with lentivirus-mediated double gene transfection, liposome-mediated double gene transfection is a relatively simple method that has lower dependency to experimental equipment and lower cost. However, the infection efficiency is significantly lower than that of lentivirus-mediated double gene transfection. The target gene continues to express after lentivirus-mediated double gene transfection, and no gene loss occurs after subculture. Figures and Tables | References | Related Articles | Metrics

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A 3D-printed calcium polyphosphate/icariin bone scaffold induces osteogenic differentiation of bone marrow mesenchymal stem cells for treating bone defects Shi Yongxin, Pang Zengjin, Yang Mingzhi, Peng Lijun, Li Fuqin, Song Hui, Luo Fuwei 2019, 23 (21):  3309-3315.  doi: 10.3969/j.issn.2095-4344.1758 Abstract ( 288 )   PDF (1011KB) ( 203 )   Save BACKGROUND: With the rapid development of tissue engineering, seeking for optimal scaffold materials, osteogenic factors and abundant stem cells with strong differentiation and proliferation potential has been an issue of concern in the field of orthopedics. OBJECTIVE: To explore the effect of calcium polyphosphate/icariin bone scaffold to induce osteogenic differentiation of bone marrow mesenchymal stem cells for the treatment of bone defects. METHODS: The CPP/ICA composite bone scaffold was prepared using 3D printing technology. The rabbit bone marrow mesenchymal stem cells were isolated and cultured. Bone defect models were made in 36 New Zealand white rabbits, and equally randomized into control group and experimental group, followed by implantation of calcium polyphosphate/icariin composite bone scaffold. Afterwards, in the experimental group, 1.5 mL of bone marrow mesenchymal stem cells (1×109/L) was injected through the femoral vein into the rabbits, while the rabbits in the control group were given the same amount of normal saline via the femoral vein. At 4, 8 and 12 weeks postoperatively, six rabbits from each group were taken to isolate and culture bone marrow mesenchymal stem cells. Transwell chamber assay was used to measure the migration ability of bone marrow mesenchymal stem cells. Fluorescence quantification-reverse transcriptase polymerase chain reaction was used to determine the type I collagen and CD44 mRNA levels in the cells. Rabbit femoral condyle specimens were taken, and X-ray films were used to observe the formation of epiphyses around the composite scaffold. Van-Gieson staining was used to observe the histological changes of bone tissues. Immunohistochemical staining was used to determine the level of epiphyseal nerve growth factors. RESULTS AND CONCLUSION: The number of migrated bone marrow mesenchymal stem cells was significantly higher in the experimental group than the control group (P < 0.05). The type I collagen and CD44 mRNA levels in the cells were also higher in the experimental group than the control group (P < 0.05). Findings from X-ray examination and histological observation showed more osteophytes in the experimental group than the control group. The level of epiphyseal nerve growth factors in the experimental group was significantly higher than that in the control group (P < 0.05). To conclude, the calcium polyphosphate/icariin bone scaffold prepared by 3D printing technology can induce the osteogenic differentiation of bone marrow mesenchymal stem cells, and has a good effect in the treatment of bone defects. Figures and Tables | References | Related Articles | Metrics

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Bone marrow mesenchymal stem cell exosomes alleviate oxygen-glucose deprivation/reperfusion injury in hippocampal neurons Shi Jiang, Gao Shilun, Liu Jinduo, Gu Tianxiang, Shi Enyi 2019, 23 (21):  3316-3322.  doi: 10.3969/j.issn.2095-4344.1749 Abstract ( 502 )   PDF (863KB) ( 188 )   Save BACKGROUND: Increasing attentions have been focused on postoperative neurological complications after deep hypothermic circulatory arrest. The core mechanism of neurological complications is global ischemia-reperfusion injury. OBJECTIVE: To investigate the protective effect and mechanism of bone mesenchymal stem cell exosomes on oxygen-glucose deprivation/reperfusion injury in hippocampal neurons. METHODS: Primary cultured fetal rat hippocampal neurons (7 days) were divided into control group, oxygen-glucose deprivation/reperfusion group (OGD/R group) and oxygen-glucose deprivation/reperfusion with adding mesenchymal stem cell exosomes (exosomes group). After 2 hours of oxygen-glucose deprivation/reperfusion, the hippocampal neurons in the latter two groups were cultured under normal conditions. After modeling, hippocampal neurons in all the three groups were cultured in nerve cell culture medium, followed by addition of bone marrow mesenchymal stem cell exosomes in the exosomes group, and addition of PBS in the other two groups. After 24 hours of continuous culture, western blot assay was used; after 48 hours of culture, the survival rate and the growth of neurites in hippocampal neurons were counted and observed by immunofluorescence staining. RESULTS AND CONCLUSION: Compared with the control group, the survival rate of hippocampal neurons in the OGD/R group was significantly reduced, the length of neurites and the number of branches were significantly impaired, and the expression level of pro-inflammatory factors was significantly increased. Compared with the OGD/R group, the survival rate of hippocampal neurons, the length of neuritis, and the number of branches in the exosomes group were significantly increased in the exosomes group, the expression levels of hepatocyte growth factor and brain-derived neurotrophic factor significantly increased, and the expression of pro-inflammatory factors obviously decreased. The results suggest that exosomes from bone marrow mesenchymal stem cells can alleviate oxygen-glucose deprivation/reperfusion injury in hippocampal neurons. The mechanism may be related to inhibiting secretion of pro-inflammatory factors secretion and promoting expression of hepatocyte growth factor and brain-derived neurotrophic factor. Figures and Tables | References | Related Articles | Metrics

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MicroRNA-431 effects on the differentiation and proliferation of human dental pulp stem cells Wu Wenhui, Guo Lei, Jiang Cuixia, Yang Yuling, Yuan Dongdong, Wang Nan 2019, 23 (21):  3323-3328.  doi: 10.3969/j.issn.2095-4344.0890 Abstract ( 235 )   PDF (715KB) ( 117 )   Save BACKGROUND: Dental pulp stem cells have great clinical potential in dental repair and reconstruction through modern cellular and molecular biology and tissue engineering approaches. OBJECTIVE: To explore the effect of microRNA-431 (miR-431) on the odontogenetic differentiation and in vitro proliferation of dental pulp stem cells. METHODS: Dental pulp stem cells were isolated by enzyme digestion and induced to odontogenetic differentiation. Alkaline phosphatase activity was measured by Alkaline Phosphatase Assay Kit. Expression of odontogenetic differentiation markers, dentin sialophosphoprotein, osteocalcin and bone sialoprotein, was detected by western blot. Expression of miR-431 was detected by qRT-PCR. Cell proliferation and colony formation ability of dental pulp stem cells were detected by MTT and colony formation assay, respectively. RESULTS AND CONCLUSION: Alkaline phosphatase activity and the expression of dentin sialophosphoprotein, osteocalcin and bone sialoprotein were increased but the expression of miR-431 reduced during the odontogenetic differentiation. Odontogenetic differentiation was inhibited by miR-431 overexpression, while miR-431-knockdown promoted the odontogenetic differentiation of dental pulp stem cells. These results indicate that miR-431 negatively regulates the odontogenetic differentiation of dental pulp stem cells, and it also inhibits the proliferation and colony formation ability of dental pulp stem cells. Figures and Tables | References | Related Articles | Metrics

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MicroRNA-20 regulates directional differentiation of stem cells from human exfoliated deciduous teeth Deng Xuan, Yang Fang, Tang Xiqing, Zhao Yan 2019, 23 (21):  3329-3335.  doi: 10.3969/j.issn.2095-4344.1761 Abstract ( 382 )   PDF (892KB) ( 156 )   Save BACKGROUND: In recent years, microRNA-20 (miR-20) has been found to plays an important role in promoting the osteogenic differentiation of bone marrow mesenchymal stem cells and inflammatory periodontal ligament stem cells. However, whether it can regulate the differentiation of dental pulp stem cells into neurons has not been fully clarified. OBJECTIVE: To investigate the mechanism by which miR-20 regulates the differentiation of stem cells from human exfoliated deciduous teeth towards neuronal cells through bone morphogenetic protein signaling pathway.  METHODS: Stem cells from human exfoliated deciduous teeth were separated and purified. Cell morphology was observed during cell induction and differentiation. Nestin, neuron-specific enolase, miR-20 and bone morphogenetic protein-2 expressions were detected. The stem cells from human exfoliated deciduous teeth were divided into five groups: negative control group (transfected with negative control sequence), miR-20 mimic group (transfected with miR-20 mimic for 48 hours), miR-20 inhibitor group (transfected with miR-20 inhibitor for 48 hours), pathway inhibition group (treated with bone morphogenetic protein pathway inhibitor for 10 days), and combined treatment group (transfected with miR-20 mimic for 24 hours and then treated with bone morphogenetic protein pathway inhibitor for 10 days). Subsequently, the expressions of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, Nestin and neuron-specific enolase were detected using real-time PCR and western blot assay at 12 days of neuronal induction. Immunofluorescence was adopted to determine the expression of III β-tubulin. Golgi staining was performed to detect the number of dendritic spines in neurons.  RESULTS AND CONCLUSION: The stem cells from human exfoliated deciduous teeth were gradually differentiated into neuron-like cells. During the differentiation period, the expression of Nestin was increased and then decreased, but the expression levels of neuron-specific enolase, miR-20 and bone morphogenetic protein-2 were gradually increased. Compared with the negative control group, miR-20 overexpression up-regulated the levels of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, III β-tubulin, Nestin and neuron-specific enolase as well as induced generation of neuronal dendritic spines. But inhibition of miR-20 or bone morphogenetic protein pathway down-regulated the protein expression of bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, III β-tubulin, Nestin and neuron-specific enolase and inhibited the generation of dendritic spines in neurons. At the same time, bone morphogenetic protein inhibitor reversed the inducement of stem cells from human exfoliated deciduous teeth to neurons caused by miR-20. Thus, miR-20 can induce the differentiation of stem cells from human exfoliated deciduous teeth towards neuron-like cells through activating the bone morphogenetic protein signaling pathway. Figures and Tables | References | Related Articles | Metrics

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Three-dimensional dynamic culture of auricular chondrocytes with microcarriers in vitro Sun Heng, Ma Shize, Jiang Haiyue, Liu Xia, Teng Li 2019, 23 (21):  3336-3341.  doi: 10.3969/j.issn.2095-4344.1755 Abstract ( 453 )   PDF (728KB) ( 114 )   Save BACKGROUND: Chondrocytes are the most commonly used seed cells in cartilage tissue engineering. How to expand chondrocytes and maintain the cell phenotype in vitro has not been solved. OBJECTIVE: To clarify the effects of three-dimensional dynamic culture on the proliferation and differentiation of chondrocytes through microcarrier culture system, providing a technical reference for the establishment of large-scale amplification methods. METHODS: Auricular chondrocytes were isolated and purified from swine ear using trypsin and collagenase digestion methods. The chondrocytes were divided into three groups: monolayer culture in petri dish, three-dimensional static culture with microcarrier, and three-dimensional dynamic culture with microcarrier. Growth of chondrocytes on the microcarrier was observed by using inverted and scanning electron microscopes. Proliferation rate of chondrocytes was measured by DNA quantitative examination method. Expression of cartilage-related genes was measured by real-time PCR. RESULTS AND CONCLUSION: The passage 1 chondrocytes rapidly attached to the surface of Cultisphere G porous microcarriers. Chondrocytes proliferated faster and distributed more evenly under three-dimensional dynamic culture than static culture. Plateau-stage chondrocytes proliferating in the monolayer culture dish were observed at 4 days of culture. Monolayer-cultured chondrocytes proliferated slowly under three-dimensional static culture, while under three-dimensional dynamic culture the cells logarithmically grew at 2-10 days of culture with the growth peak reaching at 14 days of culture. In the long-term culture, chondrocytes lost phenotypes in monolayer culture and microcarrier static culture. Three-dimensional dynamic microcarrier culture could promote cell proliferation while maintaining the cartilage phenotype. Therefore, the three-dimensional culture with microcarriers and rotary cell culture system can be a feasible method to expand auricle chondrocytes in a large scale. Figures and Tables | References | Related Articles | Metrics

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Effect of concentrated growth factors on proliferation and differentiation of human gingival fibroblasts Li Xiaoju, Song Guangbao, Yang Jianzhen, Wei Bo, Wu Bin 2019, 23 (21):  3342-3348.  doi: 10.3969/j.issn.2095-4344.1768 Abstract ( 323 )   PDF (902KB) ( 115 )   Save BACKGROUND: In the blood, there are a large number of growth factors and platelet components with regeneration and repair functions, which can promote the regeneration of soft and hard tissues. OBJECTIVE: To study the effect of concentrated growth factor extract on the proliferation and osteogenic differentiation of human gingival fibroblasts and to explore the clinical application of concentrated growth factors in oral implantology. METHODS: Patients admitted at the Department of Stomatology, People’s Hospital of Longhua, Shenzhen were selected. (1) In vitro experiment: Concentrated growth factors were extracted by centrifugation, and human gingival fibroblasts were isolated and cultured. The cells were divided into four groups and cultured in complete culture media containing 0 (control), 25%, 50% and 100% concentrated growth factor extract). (2) In vivo experiment: The affected teeth were extracted followed by dental implantation. Concentrated growth factor was placed to induce the rapid healing of soft and hard tissues. Six months later, two-stage implantation was performed. The modified envelope flap was used to increase the thickness of the labial gingiva and the amount of soft tissue in the proximal and distal areas for 3 months to obtain soft tissue contours in coordination with the adjacent teeth. RESULTS AND CONCLUSION: (1) In vitro experiment results: Compared with the control group, the proliferation of human gingival fibroblasts was significantly increased by 50% and 100% concentrated growth factor extract on the 3rd day of culture. On the 14th day of culture, compared with the control group, the activity of alkaline phosphatase, the molecular weight of mineralization and the expression of osteoblast-associated genes RUNX2 and osteocalcin were significantly increased in the 100% concentrated growth factor extract group. (2) In vivo experiment results: Concentrated growth factor extract could promote bone regeneration around oral implants. There was no obvious retraction at the gingival margin, and the gingival papilla filled the space of the teeth. The bone mass around the implant increased significantly at 14 months in relative to 3 months after implantation. To conclude, concentrated growth factor extract can promote the proliferation and osteogenic differentiation of human gingival fibroblasts and the peri-implant regeneration of soft and hard tissue. Figures and Tables | References | Related Articles | Metrics

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Vascular endothelial growth factor gene modified human amnion-derived mesenchymal stem cells promote the survival of ultra-long random skin flap Jin Wenhu, Zhou Aiting, Wu Zhonghuan, Fan Zhenhai, Wang Dali, Wei Zairong 2019, 23 (21):  3349-3356.  doi: 10.3969/j.issn.2095-4344.1743 Abstract ( 338 )   PDF (1003KB) ( 129 )   Save BACKGROUND: Studies have confirmed that vascular endothelial growth factor can induce angiogenesis, and human amnion-derived mesenchymal stem cells improve the repair of ischemia-reperfusion injury using flap transplantation via various mechanisms. Therefore, the modification of human amnion-derived mesenchymal stem cells using vascular endothelial growth factor gene promotes the survival of skin flaps. OBJECTIVE: To investigate the effect of human amnion-derived mesenchymal stem cells modified by vascular endothelial growth factor gene on the survival of ultra-long random skin flaps. METHODS: Thirty healthy adult Sprague-Dawley rats were randomly divided into group I, group II and group III, 10 rats in each group. Two ultra-long ischemic random skin flap models were constructed at the left and right back of each rat, and the skin flap pedicle was located at iliac spine level. These skin flaps received the following treatment immediately after removal: The flaps were divided into group I-left, I-right; II-left, II-right; III-left and III-right. 0.5 mL of LG-DMEM was injected in the skin flaps in I-left and II-left groups (control group); 0.5 mL of 1×109/L human amnion-derived mesenchymal stem cells was injected into the skin flaps in I-right and III-left groups (experimental group 1); 0.5 mL of 1×109/L vascular endothelial growth factor-transfected human amnion-derived mesenchymal stem cells was injected into the flaps in II-right and III-right groups (experimental group 2). There were five injection sites for each flap, 0.1 mL per each site. The blood perfusion in the middle and pedicle of each skin flap was monitored using laser Doppler flow meter immediately, 1, 2, 4 and 7 days after transplantation. The survival rate of skin flaps in all groups was measured at 7 days after transplantation. The capillary density in survived skip flaps was histologically observed. The distribution and survival rate of CM-Dil labeled human amnion-derived mesenchymal stem cells were observed by fluorescent microscope. The vascular endothelial growth factor protein level was detected by western blot. RESULTS AND CONCLUSION: (1) The survival rate of skin flaps, blood perfusion, and capillary density showed significantly difference among groups (P < 0.05) and ranked as follows: experimental group 2 > experimental group 1 > control group. (2) Under fluorescent microscope, CM-Dil labeled human amnion-derived mesenchymal stem cells were visible in the flaps in the experimental groups 1 and 2. (3) Vascular endothelial growth factor positively expressed in all the flaps, while the protein expression of vascular endothelial growth factor was ranked as follows: experimental group 2 > experimental group 1 > control group and there were significant differences among groups (P < 0.05). To conclude, the application of human amnion-derived mesenchymal stem cells in the middle and distal parts of ultra-long random skin flap can remarkably improve the survival rate of skin flaps, and moreover transfection with vascular endothelial growth factor can increase the secretion of vascular endothelial growth factor, further improving the effects of human amnion-derived mesenchymal stem cells promoting angiogenesis and survival rate of ultra-long random skip flaps. Figures and Tables | References | Related Articles | Metrics

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Effects of mesenchymal stem cell conditioned media from different sources on intrinsic aging cells Zheng Guichun, Zhao Shucan, Huang Lizhen, Zhang Xiaofang, Wang Bingyun, Chen Shengfeng, Chen Zhisheng 2019, 23 (21):  3357-3363.  doi: 10.3969/j.issn.2095-4344.1765 Abstract ( 528 )   PDF (736KB) ( 162 )   Save   BACKGROUND: With the economic development, people have an increasing pursuit of beauty, and anti-aging research has become a hot research topic. Stem cells have self-renewal and multi-directional differentiation ability, and mesenchymal stem cells are one of them. Studies have shown that mesenchymal stem cells can repair aging cells through paracrine action, but little has been reported on the anti-aging effects of mesenchymal stem cell conditioned media from different sources. OBJECTIVE: To compare the anti-aging effects of human adipose-, amniotic fluid-, placenta- and umbilical cord-derived mesenchymal stem cell conditioned media on aging skin fibroblasts.  METHODS: Human adipose, amniotic fluid, placenta and umbilical cord-derived mesenchymal stem cell conditioned media were prepared and used to culture aging skin fibroblasts. Cell viability was detected by cell counting kit-8 and cell senescence rate measured by β-galactosidase staining kit. DCFH-DA probe was used to detect the content of reactive oxygen species. qPCR was used to detect the expression levels of p16, p53, COL1 and KLOTHO genes.  RESULTS AND CONCLUSION: (1) Four sources of mesenchymal stem cell conditioned media could increase the cell viability of skin fibroblasts, among which the adipose-derived mesenchymal stem cell conditioned medium had the best effect and the lowest β-galactosidase staining rate. (2) Four sources of mesenchymal stem cell conditioned media could reduce reactive oxygen species content, and adipose- and amniotic fluid-derived mesenchymal stem cell conditioned media had the lowest content of reactive oxygen species. (3) p16 and p53 levels in the placenta and umbilical cord-derived mesenchymal stem cell conditioned media were higher than those in the control group. The expression of p53 in the amniotic fluid-derived mesenchymal stem cell conditioned medium was lower than that in the control group, but there was no difference in the p16 expression between the two groups. Compared with the control group, the expression of p16 was lower and the expression of p53 was higher in the adipose-derived mesenchymal stem cell conditioned medium. (4) Compared with the control group, the expression of COL1 gene was significantly higher in the different culture media except for the amniotic fluid-derived mesenchymal stem cell conditioned medium. Compared with the control group, the expression of KLOTHO was significantly higher in different culture media except for placenta-derived mesenchymal stem cell conditioned medium, and highest in the adipose-derived mesenchymal stem cell conditioned medium. To conclude, mesenchymal stem cell conditioned media from four different sources can delay cell senescence to a certain extent, though its mechanism remains to be further studied. Figures and Tables | References | Related Articles | Metrics

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Isolation, identification and chondrogenic differentiation of rat articular cartilage-derived progenitor cells Shen Xu, Lu Yingjie, Lu Dongdong, Fang Yuepeng, Zhou Naihui, Zhu Xuesong, Zhu Yueqian 2019, 23 (21):  3364-3370.  doi: 10.3969/j.issn.2095-4344.1756 Abstract ( 498 )   PDF (858KB) ( 210 )   Save BACKGROUND: Articular cartilage-derived progenitor cells have strong chondrogenic differentiation ability and limited osteogenic differentiation ability. However, it is unclear whether and how the cellular characteristics of articular cartilage-derived progenitor cells after amplification change. OBJECTIVE: To observe the proliferation, osteogenesis and chondrogenesis ability of articular cartilage-derived progenitor cells in different generations as compared with bone marrow mesenchymal stem cells. METHODS: Articular cartilage-derived progenitor cells were sorted from chondrocytes by using differential adhesion to fibronectin. Cell counting kit-8 and colony formation assay were used to observe the proliferative capacity of articular cartilage-derived progenitor cells. Osteogenic differentiation potential of articular cartilage-derived progenitor cells and bone marrow mesenchymal stem cells were measured by alizarin red staining and determination of osteogenic gene expression. Collagen II immunohistochemical staining and determination of chondrogenic-related gene expression were used to measure the change in the chondrogenic ability of articular cartilage-derived progenitor cells and bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION: Articular cartilage-derived progenitor cells sorted by fibronectin had strong proliferation ability, osteogenic and chondrogenic differentiation potential. The results of cell counting kit-8 and colony formation assay showed that articular cartilage-derived progenitor cells had a slight decrease in cell proliferation during cell expansion. Bone marrow mesenchymal stem cells presented with strong osteogenic potential throughout cell expansion, while the chondrogenic potential of the cells decreased gradually during cell expansion. Articular cartilage-derived progenitor cells had strong chondrogenic potential with no considerable change during cell expansion, while the osteogenic potential of the cells decreased gradually significantly during cell expansion. To conclude, the osteogenic potential of articular cartilage-derived progenitor cells was significantly lower than that of bone marrow mesenchymal stem cells, and it gradually decreased during cell expansion. The chondrogenic potential of articular cartilage-derived progenitor cells was higher than that of bone marrow mesenchymal stem cells, but there was no significant change during cell expansion. These findings indicate that articular cartilage-derived progenitor cells may be an ideal kind of seed cells for cartilage tissue engineering. Figures and Tables | References | Related Articles | Metrics

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Effects of magnesium on proliferation, adhesion and apoptosis of endothelial progenitor cells in patients with preeclampsia Li Li, Wang Xinling, Liu Suxin, Huo Yan 2019, 23 (21):  3371-3377.  doi: 10.3969/j.issn.2095-4344.1754 Abstract ( 336 )   PDF (796KB) ( 128 )   Save BACKGROUND: Magnesium ions can reduce the sensitivity of vascular endothelial cells to adverse stimuli and regulate vascular endothelial function by scavenging free radicals, stabilizing cell membranes and inhibiting inflammation. However, the effect of magnesium ions on endothelial progenitor cells is still unclear. OBJECTIVE: To explore the effects of magnesium on the proliferation, adhesion and apoptosis of endothelial progenitor cells in patients with preeclampsia. METHODS: From March 2016 to October 2018, 17 preeclampsia pregnant patients and 15 normal pregnant women who were admitted in the Obstetrics Department of Hebei General Hospital were enrolled. Endothelial progenitor cells were isolated and cultured from umbilical cord blood in the two groups of pregnant women. Endothelial progenitor cells isolated from each preeclampsia pregnant patient were divided into four groups: preeclampsia group was intervened with PBS, and low, middle and high concentration magnesium ion groups were intervened with the medium containing 4, 8, and 16 mmol/L magnesium ions, respectively. Endothelial progenitor cells from normal pregnant women were treated with PBS as control group. After 24 hours of intervention, cell proliferation, adhesion, apoptosis, inflammation (interleukin-6, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein-1 levels) and oxidative stress (superoxide dismutase and malondialdehyde levels) were detected. The study protocol was approved by the Ethics Committee of Hebei General Hospital with the approval No. 2013linshen13. RESULTS AND CONCLUSION: (1) Compared with the control group, the cell proliferation and adhesion of the preeclampsia group were significantly decreased (P < 0.05); the apoptotic rate significantly increased (P < 0.05); the expression of Bax and Active Caspase-3 increased, and the expression of Bcl-2 decreased (both P < 0.05); the levels of interleukin-6, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, and malondialdehyde increased (P < 0.05), and the activity of superoxide dismutase decreased (P < 0.05). (2) Compared with the preeclampsia group, the cell proliferation and adhesion of the magnesium ion intervention groups significantly increased (P < 0.05); the apoptotic rate decreased (P < 0.05); the expression of Bax and Active Caspase-3 decreased, and the expression of Bcl-2 increased (P < 0.05); the levels of interleukin-6, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, malondialdehyde decreased (P < 0.05), and the level of superoxide dismutase increased (P < 0.05). And the improvement in the high concentration magnesium ion group was better than that in the low and middle concentration magnesium ion groups (P < 0.05). To conclude, magnesium ions can promote the proliferation and adhesion of endothelial progenitor cells in vitro, inhibit the secretion of inflammatory factors, and reduce the apoptotic rate and oxidative stress level in patients with preeclampsia. Figures and Tables | References | Related Articles | Metrics

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Basic fibroblast growth factor-transfected bone marrow mesenchymal stem cell transplantation for chronic obstructive pulmonary disease Wu Huala, Zhou Xiangxiang, Zhong Yulan, Gan Xin 2019, 23 (21):  3378-3385.  doi: 10.3969/j.issn.2095-4344.1750 Abstract ( 283 )   PDF (975KB) ( 130 )   Save BACKGROUND: The imbalance between pro-inflammatory and anti-inflammatory responses plays a key role in the pathogenesis of chronic obstructive pulmonary disease. The research team attempted to cure the chronic obstructive pulmonary disease by reducing the intrapulmonary inflammatory response, promoting the regeneration and repair of alveolar epithelial or bronchial epithelial cells, and alleviating or reversing the pathological procession of the disease. OBJECTIVE: To observe the expression of interleukin-10 and interleukin-4 after transplantation of basic fibroblast growth factor (bFGF)-transfected bone marrow mesenchymal stem cells (BMSCs) and cell differentiation in rats with chronic obstructive pulmonary disease. METHODS: BMSCs were isolated and cultured using the whole bone marrow adhesion method. 150 healthy Sprague-Dawley rats were randomly selected, 30 of which were randomly selected as normal controls and the remaining rats were used to make a chronic obstructive pulmonary disease model by lipopolysaccharide combined with fumigation. Model rats were then randomly divided into chronic obstructive pulmonary disease group (model group), BMSCs group, pcDNA3.1-BMSCs group, and bFGF-pcDNA3.1-BMSCs group (n=30 per group). The rats in each group were sacrificed at 7, 14 and 28 days. The pathological changes of the lung tissues were observed by hematoxylin-eosin staining. The levels of interleukin-10 and interleukin-4 in the peripheral blood were detected by ELISA. The mRNA levels of interleukin-10 and interleukin-4 in the lung tissues were detected by qRT-PCR. The differentiation of BMSCs in the lung tissues was detected by immunofluorescence staining. RESULTS AND CONCLUSION: On the 28th day after modeling, the pathological changes of the lung tissues were significantly improved in the three BMSCs groups relative to the model group. The expressions of interleukin-10 and interleukin-4 in peripheral blood and lung tissues at the same observation time point were significantly higher in the three BMSCs groups than the model group (P < 0.05). Although there was no significant difference between the BMSCs and pcDNA3.1-BMSCs group, higher levels of interleukin-10 and interleukin-4 were detected in the bFGF-pcDNA3.1-BMSCs group than the other BMSCs groups (P < 0.05). On the 28th day after modeling, some cells in the lung tissues in the three BMSCs groups were concurrently positive for CM-Dil and SPC or CC16. Moreover, there were more positive cells in the bFGF-pcDNA3.1-BMSCs group as compared with the other BMSCs groups. In conclusion, transplantation of bFGF-transfected BMSCs for chronic obstructive pulmonary disease in rats can reduce pathological changes of the lung tissue, enhance anti-inflammatory effects, and promote differentiation of BMSCs into alveolar epithelial cells and bronchial epithelial cells. Figures and Tables | References | Related Articles | Metrics

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Repairing hypoxic-ischemic brain damage in neonatal rats by nasal transplantation of human umbilical cord mesenchymal stem cells Yang Hanhua, Tian Shufeng, Xie Lichun, Chen Yunbin, Ma Lian 2019, 23 (21):  3386-3391.  doi: 10.3969/j.issn.2095-4344.1762 Abstract ( 385 )   PDF (766KB) ( 128 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells exert significant neuroprotective effects in animals, including reducing neuronal apoptosis and promoting neuronal regeneration. Current approaches to transfer these cells include transvenous, arterial, lumbar puncture, brain stereotactic transplantation and lateral ventricle transplantation, but all of them are insufficient.  OBJECTIVE: To investigate the feasibility and effectiveness of nasal transplantation of human umbilical cord mesenchymal stem cells in the treatment of hypoxic-ischemic brain damage in neonatal rats. METHODS: Ninety 7-day-old Sprague-Dawley rats (provided by the Experimental Animal Center of Shantou University Medical College) were randomly divided into three groups (n=30 per group): in sham operation group the left common carotid artery was only separated and then sutured; in model group, an animal model of hypoxic-ischemic brain damage was prepared through ligation and occlusion of the left common carotid artery combined with hypoxic chamber; in experimental group, the animal model was prepared as described in the model group, and then green fluorescent protein-labeled human umbilical cord mesenchymal stem cells were instilled into the nasal cavity of model rats. Brain tissue was taken 3 days after modeling, and Nissl staining, TUNEL detection, and immunofluorescence staining were performed. The memory and learning abilities of the rats were detected by water maze at 14 and 29 days after modeling. After 14-day water maze test, TUNEL staining of the brain tissue was performed. The animal experiment was approved by the Experimental Animal Ethics Committee of Shenzhen University Health Science Center with the approval No. 2016-121. RESULTS AND CONCLUSION: (1) Nissl staining: Neurons in the cerebral cortex and hippocampal CA1 area of the model group were irregularly arranged, and there were few Nissl bodies. Compared with the model group, the neurons in the cerebral cortex and hippocampal CA1 area were arranged more regularly in the experimental group, and there were more Nissl bodies. (2) TUNEL staining: On 3 and 14 days after modeling, the number of apoptotic cells in the model group was significantly higher than that in the sham operation group (P < 0.05) and in the experimental group (P < 0.05). (3) Immunofluorescence staining: Green-labeled human umbilical cord mesenchymal stem cells were found in the brain tissue of the experimental group, mainly distributed in the hippocampus and cerebral cortex of the injured side. Green fluorescent protein-labeled cells were mainly visible in the injured hippocampus and cortex, but rarely detected in the non-injured brain tissues. Non-GFAP or NSE double staining cells were detected. (4) The average escape latency of the experimental group was lower than that of the model group (P < 0.05). Overall, these findings indicate that nasal transplantation of human umbilical cord mesenchymal stem cells can reduce neuronal apoptosis and improve memory and learning abilities of neonatal rats with hypoxic-ischemic brain damage. Figures and Tables | References | Related Articles | Metrics

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Treating deep vein thrombosis with human umbilical cord mesenchymal stem cell transplantation Wang Yan, Shi Qin, Zhang Yuquan 2019, 23 (21):  3392-3397.  doi: 10.3969/j.issn.2095-4344.1745 Abstract ( 476 )   PDF (855KB) ( 123 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells can home to ischemic sites and form functional blood vessels locally. Neovascularization is critical for the organization and recanalization of deep vein thrombosis. However, transplantation of human umbilical cord mesenchymal stem cells for the treatment of thrombosis has not been reported. OBJECTIVE: To investigate the dissolution, organization and recanalization of deep vein thrombosis after the transplantation of human umbilical cord mesenchymal stem cells. METHODS: Deep vein thrombosis model was established by “narrow method” in rats. Suspension of the third generation human umbilical cord mesenchymal stem cells at a density of 2×109 cells/L was prepared. The model rats were randomly divided into four groups. In control group, 1 mL of PBS was injected into the tail vein on days 1, 2, and 3 after modeling. In experimental group 1, 1 mL of cell suspension was injected into the tail vein on day 1 after modeling, and 1 mL of PBS was injected on days 2 and 3. In experimental group 2, 1 mL of cell suspension was injected on days 1 and 2 after modeling, and 1 mL of PBS was injected on day 3. In experimental group 3, 1 mL of cell suspension was injected on days 1, 2, and 3 after modeling. The experimental protocol was approved by the Animal Experimental Ethics Committee of Nantong University with the approval No. 20180315-001 on April 1, 2018. Samples from deep vein thrombosis rats were taken on day 7 after modeling. Number of new blood vessels and dissolution rate in each group were detected by hematoxylin-eosin staining and immunohistochemical staining. RESULTS AND CONCLUSION: Compared with the control group, the capillary density of the experimental groups 2 and 3 was significantly increased (P < 0.01). The dissolution rate of all the experimental groups was higher than that of the control group, but the difference was not statistically significant (P > 0.05). All these results reveal that a certain amount of human umbilical cord mesenchymal stem cells transplanted can increase the number of microvessels in the thrombosis, and accelerate organization and recanalization of the thrombosis. Figures and Tables | References | Related Articles | Metrics

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Bone marrow colony forming units-fibroblast in patients with chronic myelomonocytic leukemia: functions and clinical significance Xu Ruohao, Li Chao, Wu Ping, Li Minming, Deng Chengxin, Geng Suxia, Lai Peilong, Lu Zesheng, Weng Jianyu, Du Xin 2019, 23 (21):  3398-3403.  doi: 10.3969/j.issn.2095-4344.1725 Abstract ( 443 )   PDF (908KB) ( 151 )   Save BACKGROUND: Studies have shown that abnormal bone marrow microenvironment is involved in the development of chronic myelomonocytic leukemia. Colony forming unit-fibroblast represents a group of multipotent mesenchymal stromal cells that can reflect the bone marrow stromal microenvironment in terms of cell function and number. OBJECTIVE: To investigate the abilities of bone marrow mesenchymal stromal cells to form colony forming units-fibroblast and to differentiate towards osteoblasts in patients with chronic myelomonocytic leukemia and to study the relationship between colony forming unit-fibroblast counts and clinic characteristics (peripheral blood cell count and clinical phenotype). METHODS: (1) Fifteen newly diagnosed chronic myelomonocytic leukemia patients admitted to the Department of Hematology, Guangdong Provincial People’s Hospital, and 10 volunteers (control) were enrolled. Functions and osteogenic potential of colony forming units-fibroblast were compared between two groups. (2) There was a correlation analysis between colony forming unit-fibroblast counts and clinical characteristics (peripheral blood cell count, clinical phenotype, percentage of bone marrow blasts and gene mutations). The study protocol was approved by the ethics committee of Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences) with the approval No. [2018]002. RESULTS AND CONCLUSION: (1) The number of colony forming units-fibroblast formed in the patients with chronic myelomonocytic leukemia decreased as compared with the control group (P=0.04). The 10 of 15 (67%) patients showed remarkable reduction in the colony forming unit-fibroblast counts (P=0.00), while the rest 5 (33%) patients exhibited normal colony forming unit-fibroblast levels (P=0.14). (2) Expression levels of Osterix and Runx2 in the bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia were significantly lower than those in the control group. There was a limited osteogenic potential in the bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia (P=0.00). (3) Colony forming unit-fibroblast counts of patients with chronic myelomonocytic leukemia were negatively related to peripheral white blood cells, neutrocytes and monocytes counts, and were positively related to peripheral platelet counts. (4) Nine out of 10 (90%) patients with reduced colony forming unit-fibroblast counts exhibited a higher white blood cell level (≥ 13×109/L) (P=0.01), and 5 out of 10 (50%) patients carried NRAS/KRAS mutations. Findings from this study show limited function of colony forming unit-fibroblast formation and osteogenic differentiation in bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia. Moreover, the number of colony forming units-fibroblast seems to be correlated to peripheral blood cell counts and clinical features, indicating underlying significances in the diagnosis and prognosis of chronic myelomonocytic leukemia. Figures and Tables | References | Related Articles | Metrics

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Effects of microRNA-9 on proliferation and migration of thyroid papillary cancer stem cells in vitro Han Fei, Qin Haixia 2019, 23 (21):  3404-3409.  doi: 10.3969/j.issn.2095-4344.1763 Abstract ( 332 )   PDF (721KB) ( 155 )   Save BACKGROUND: MicroRNA-9 (miR-9) exerts different effects in different types of tumors and at different stages of tumorigenesis, but its role in thyroid papillary carcinoma remains unclear. OBJECTIVE: To analyze the effects of miR-9 on the proliferation and migration of thyroid papillary cancer stem cells in vitro. METHODS: ALDH1 positive cells and ALDH1 negative cells were separated from human papillary thyroid cancer cells by flow cytometry. Expression of miR-9 mRNA was detected using reverse transcription PCR. ALDH1 positive cells in logarithmic growth phase were transfected with miR-9 mimetic, miR-9 inhibitor or miR mimetic control. After 48 hours, the transfection efficiency of miR-9 was detected by qRT-PCR; cell proliferation, apoptosis and migration were detected by MTT, TUNEL, and Transwell chamber assay, respectively. The expression of apoptosis-related proteins (Bcl-2 and Bax) and migration-related proteins (matrix metalloproteinase-2 and matrix metalloproteinase-9) was detected by western blot. RESULTS AND CONCLUSION: Successful transfections were confirmed by increased miR-9 gene expression in the miR-9 mimetic group and decreased miR-9 expression in the miR-9 inhibitor group relative to the miR mimetic control group (P < 0.05). Compared with the miR mimetic control group, the miR-9 mimetic group showed decreased cell proliferation and migration ability, and increased apoptosis, while in the miR-9 inhibitor group, cell proliferation and migration abilities were increased, and cell apoptosis decreased. Compared with the miR mimetic control group, the expression of Bcl-2 protein was decreased and the expression of Bax protein increased in the miR-9 mimetic group (P < 0.05), and the expression of Bcl-2 protein increased and expression of Bax protein decreased in the miR-9 inhibitor group (P < 0.05). Compared with the miR mimetic control group, the expression of matrix metalloproteinase 2 and matrix metalloproteinase-9 was decreased in the miR-9 mimetic group (P < 0.05), and the expression of matrix metalloproteinase 2 and matrix metalloproteinase-9 in the miR-9 inhibitor group was increased (P < 0.05). To conclude, miR-9 can inhibit the proliferation and migration of thyroid papillary cancer stem cells in vitro, but increase cell apoptosis. Figures and Tables | References | Related Articles | Metrics

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Interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha inhibit expression of type II collagen in nucleus pulposus cells Liang Weidong, Ren Zhouliang, Sheng Jun, Cao Rui, Sheng Weibin 2019, 23 (21):  3410-3417.  doi: 10.3969/j.issn.2095-4344.1748 Abstract ( 414 )   PDF (1131KB) ( 137 )   Save BACKGROUND: The role of inflammatory factors in the degeneration of intervertebral discs has received increasing attention. The mechanism by which inflammatory factors inhibit the expression of type II collagen has not been fully elucidated. OBJECTIVE: To observe the expression of type II collagen in nucleus pulposus cells after intervention with inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and the mechanism of action. METHODS: Adult male Sprague-Dawley rats were provided by the Animal Experimental Center of Xinjiang Medical University, and the study protocol was approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval No. IACUC20151203-09). Nucleus pulposus cells from rats were isolated, cultured, identified and randomly divided into seven groups: control group, IL-1β (50 μg/L) group, IL-6 (100 μg/L) group, TNF-α (20 μg/L) group, IL-1β (50 μg/L)+740Y-P (a PI3K activator) group, IL-6 (100 μg/L)+ 740Y-P group, TNF-α (20 μg/L)+740Y-P group. After 48 hours of intervention, nucleus pulposus cells were collected. RT-PCR, western blot and immunofluorescence staining were used to detect the effects of IL-1β, IL-6 and TNF-α on expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase, type I and type II collagens, Aggrecan, and PI3K/AKT signaling pathway proteins in nucleus pulposus cells. RESULTS AND CONCLUSION: (1) The expression of tissue inhibitor of metalloproteinase-1 mRNA in IL-1β and TNF-α groups was significantly lower than that in the control group (P < 0.05). The mRNA levels of matrix metalloproteinases-9 and -13 in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group (P < 0.05). The expression of IL-6 mRNA in all experimental intervention groups was significantly higher than that in the control group (P < 0.05). The mRNA levels of IL-1β, IL-6 and TNF-α in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group. The expression of interleukin-1ra in the IL-1β and TNF-α groups was significantly lower than that in the control group (P < 0.05). (2) Immunofluorescence results showed that compared with the control group, the expression of type II collagen and Aggrecan significantly decreased in the IL-1β and TNF-α groups, and slightly decreased in the IL-6 group. Compared with IL-1β, IL-6 and TNF-α groups, the expression of type II collagen and Aggrecan was significantly increased in the three corresponding PI3K activator groups. These findings indicate that IL-1β, IL-6 and TNF-α as inflammatory factors may inhibit the expression of type II collagen in nucleus pulposus cells by inhibiting the PI3K/AKT cell proliferation pathway. Figures and Tables | References | Related Articles | Metrics

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Expression profile analysis of miRNAs in serumal exosomes as sensitive biomarkers in patients with graft-versus-host disease following allogeneic hematopoietic stem cell transplantation Liu Jiao, Wang Daming, An Taixue, Hu Xuesong, Li Nankai, Wang Hongfu, Ma Wen, Nie-He Zhongrong, Xiao Lijia, Zhou Yiwen, Zheng Lei 2019, 23 (21):  3418-3425.  doi: 10.3969/j.issn.2095-4344.1764 Abstract ( 359 )   PDF (962KB) ( 155 )   Save BACKGROUND: There are few studies, especially in China, addressing the correlation between exosome miRNAs and graft-versus-host disease (GVHD) following human hematopoietic stem cell transplantation. Exosome-derived nucleic acid molecules are expected to become new biomarkers for predicting the occurrence of acute GVHD (aGVHD). OBJECTIVE: To investigate the differential expression of candidate gene microRNAs in peripheral blood serum exosomes of aGVHD patients with after allogeneic hematopoietic stem cell transplantation.  METHODS: A total of 30 patients with aGVHD and without aGVHD after allogeneic hematopoietic stem cell transplantation from August 2017 to May 2018 were enrolled, including 20 in aGVHD group and 10 in non-aGVHD group. Serum exosomes and miRNAs were extracted before and after transplantation. Image acquisition and data analysis were performed after gene chip hybridization.  RESULTS AND CONCLUSION: After microarray hybridization microRNAs showed an over 10-fold upward or downward trend before and after transplantation. Compared with the control group, 11 microRNAs increased and 26 decreased in the aGVHD group. hsa-miR-3976, hsa-miR-122-5p, and hsa-miR-3125 were significantly up-regulated, while hsa-miR-941, hsa-miR-4687-5P and hsa-miR-4769-5p were significantly down-regulated. Figures and Tables | References | Related Articles | Metrics

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Proanthocyanidins inhibit high glucose-induced apoptosis in nucleus pulposus cells in a rabbit Xu Qing, Fang Haolin, Liu Yang, Zhang Cunxin, Tian Baofang 2019, 23 (21):  3426-3431.  doi: 10.3969/j.issn.2095-4344.1747 Abstract ( 361 )   PDF (845KB) ( 133 )   Save BACKGROUND: Proanthocyanidins are an internationally recognized active oxygen free radical scavenger in the human body due to their high antioxidant activity. OBJECTIVE: To investigate whether proanthocyanidins has a significant inhibitory effect on high glucose-induced apoptosis in nucleus pulposus cells and to investigate its molecular mechanism. METHODS: Healthy 2-month-old New Zealand white rabbits were provided by Shandong Kond Biotechnology Co., Ltd., and the study protocol was approved by Health Care Technology Association of Shandong Province, China. Nucleus pulposus cells were isolated, cultured and passaged to the 5 generation in vitro. Passage 3 nucleus pulposus cells at a concentration of 1×108/L were inoculated into six 4-well culture plates and cultured in proanthocyanidins at different concentrations (0, 10, 20, 30, 40, 50, 60 µmol/L). Cell proliferation was detected using cell counting kit-8, to determine the proper concentration of proanthocyanidins. Cells were divided into four groups and cultured normally, in the medium containing 100 mmol/L glucose, in the medium containing 40 µmol/L proanthocyanidins, and in the medium containing 40 µmol/L proanthocyanidins and 100 mmol/L glucose (combination group, in which 100 mmol/L glucose was added after 6-hour pretreatment with 40 µmol/L proanthocyanidins), respectively. The morphology of nucleus pulposus cells was observed by inverted microscope, and the cell growth curve was drawn. The apoptotic rate, intracellular reactive oxygen species and expression of heme oxygenase in the cells were detected. RESULTS AND CONCLUSION: Nucleus pulposus cells from rats showed polygonal and short spindle shapes and cellular pseudopod appeared gradually and then became slim with increasing passage numbers. The cells grew in an “S”-shaped curve. The cells grew slowly on day 1 after passage, and grew rapidly from day 2 to day 5. As the number of passages increased, the cell viability gradually decreased. It was found that the proper concentration of proanthocyanidins was 40 μmol/L. Flow cytometry results showed that the apoptosis rate of nucleus pulposus cells in the high glucose group was significantly increased. Pretreatment with proanthocyanidins significantly decreased the apoptosis rate as compared to the high glucose group (P < 0.05). Results of western blot assay showed that proanthocyanidins significantly increased the expression of heme oxygenase-1, and moreover, the expression of heme oxygenase 1 was negatively correlated with the apoptotic rate, active oxygen free radical accumulation and cell proliferation activity. All the findings indicate that proanthocyanidins could inhibit the apoptosis of nucleus pulposus cells induced by high glucose, and decrease intracellular reactive oxygen species accumulation and proliferation activity by up-regulating the expression of heme oxygenase 1. Figures and Tables | References | Related Articles | Metrics

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Biological characteristics of menstrual blood-derived mesenchymal stem cells and their clinical application in tissue repair and reconstruction Chang Qiyuan, Tan Jichun 2019, 23 (21):  3432-3438.  doi: 10.3969/j.issn.2095-4344.1746 Abstract ( 359 )   PDF (684KB) ( 145 )   Save BACKGROUND: Mesenchymal stem cells have great application potentials in immune regulation and tissue repair. Recently, menstrual blood-derived mesenchymal stem cells have attracted the attention of researchers because of their advantages, including easy acquisition, low immunogenicity and no ethical restrictions. OBJECTIVE: To review the biological characteristics of menstrual blood-derived mesenchymal stem cells and their application in multiple systems and diseases. METHODS: Relevant articles published from 2000 to 2018 were searched in PubMed, CNKI and Google Scholar using the keywords of “endometrium; menstrual blood; mesenchymal stem cells” in English and Chinese, respectively.  RESULTS AND CONCLUSION: Menstrual blood-derived mesenchymal stem cells have a strong potential in self-renewal and multilineage differentiation. These cells regulate immune responses, accelerate angiogenesis, and promote tissue repair. The use of menstrual blood-derived mesenchymal stem cells has varying degrees of achievements in the immune and reproductive systems and metabolic diseases. Mechanisms of action underlying menstrual blood-derived mesenchymal stem cells are still insufficient. Further investigations on the paracrine effect of menstrual blood-derived mesenchymal stem cells will be of great significance for the treatment of traumatic and degenerative diseases, angiogenesis and tissue repair. Clinical trials regarding some crucial issues, such as safety of menstrual blood-derived mesenchymal stem cells, duration of immunomodulation and long-term cell effects in the human body, are warranted in the future. Figures and Tables | References | Related Articles | Metrics

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Application and potential of stem cells in the repair of facial nerve injury Xiao Guangqing, Lun Zhigang, Chen Huizhen, Li Aimin 2019, 23 (21):  3439-3444.  doi: 10.3969/j.issn.2095-4344.1724 Abstract ( 434 )   PDF (691KB) ( 114 )   Save BACKGROUND: Facial nerve injury is extremely common, and autologous nerve transplantation as its “gold standard” treatment has limitations. Therefore, stem cell therapy for facial nerve injury has become a hot topic in recent years. OBJECTIVE: To elaborate the progress in basic experiments of stem cells in the repair of facial nerve injury, by describing the characteristics and applications of experimental animal models, tissue engineering and various stem cells. METHODS: PubMed, CNKI, and WanFang databases were retrieved for relevant articles published from 2005 to 2018. The keywords were “stem cell, tissue engineering, facial nerve” in Chinese and English. Literature regarding relevant experimental research and reviews was searched, and finally 54 articles were included. RESULTS AND CONCLUSION: Stem cells used in facial nerve injury repair mainly include neural stem cells, bone marrow mesenchymal stem cells, adipose stem cells, odontogenic stem cells, skeletal muscle stem cells, and olfactory stem cells, which are characterized by various types, wide sources, rapid proliferation and multi-directional differentiation. Stem cell therapy has great potential to replace autologous nerve transplantation and become the main treatment for facial nerve injury in the future. Figures and Tables | References | Related Articles | Metrics