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BACKGROUND:The microRNAs are involved in regulation of stem cel proliferation, differentiation and aging. To study the effect of Let-7c, a member of Let-7, on the neural differentiation of bone marrow mesenchymal stem cels provides new ideas for stem cel therapy. OBJECTIVE: To investigate the role of Let-7c in the neural differentiation of bone marrow mesenchymal stem cels. METHODS: The lentiviral vectors of Let-7c-up and Let-7c-inhibition were constructed and transfected into rat bone marrow mesenchymal stem cels. Optimal multiplicity of infection was screened. The cels were divided into non-transfected group, negative control group (transfected with empty virus), transfected enhancement group (transfected with LV-rno-Let-7c-up), transfected inhibition group (transfected with LV-rno-Let-7c-5p-inhibition). Bone marrow mesenchymal stem cels were treated with fasudil as an inducer for triggering the cels to differentiate into neurons. The fluorescence expressed by transfected cels was observed under inverted fluorescence microscope. The expression of neuron-specific markers, neuron-specific enolase and microtubule-associated protein 2, were measured by immunocytochemical method. The mRNA expression of microtubule-associated protein 2 was detected by RT-PCR. The cel viability was determined by MTT method. RESULTS AND CONCLUSION:Under the inverted fluorescence microscope, the cels were successfuly transfected with LV-rno-Let-7c-up and LV-rno-Let-7c-5p-inhibition. Fasudil induced bone marrow mesenchymal stem cels to differentiate into neurons. The transfection efficiency and expression levels of neuron-specific enolase and microtubule-associated protein 2 in the transfected enhancement group were significantly higher than those in the negative control group (P < 0.05), while in the transfected inhibition group, they were lower than those in the negative control group (P < 0.05). These findings indicate that the differentiation percentage of bone marrow mesenchymal stem cels is increased by fasudil after transfection with LV-rno-Let-7c-up, and Let-7c may promote the differentiation of bone marrow mesenchymal stem cels into neurons.
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BACKGROUND:Numerous studies have demonstrated that bone marrow mesenchymal stem cels can be induced to differentiate into myocardial cels under certain conditions. Dimethyl sulfoxide is one of the commonly used inducers, and its mechanism is mainly by inhibiting the c-myc gene expression, thus reducing endogenous poly(adenosine diphosphate nucleotide) level. OBJECTIVE:To study the feasibility of dimethyl sulfoxide inducing the myocardial differentiation of bone marrow mesenchymal stem cels and its optimal concentration. METHODS:Bone marrow mesenchymal stem cels from Sprague-Dawley rats were isolated and culturedin vitro, and then induced by dimethyl sulfoxide to differentiate into myocardial cels. According to the concentrations of dimethyl sulfoxide, there were three groups: 0.6%, 0.8% and 1.0% group. Additionaly, a blank control group with no induction was set up. After 72 hours of induction, induction media were removed, and cels were then cultured in normal media for 4 weeks. RESULTS AND CONCLUSION: Morphology and immunocytochemistry detection results confirmed that dimethyl sulfoxide could induce the differentiation of bone marrow mesenchymal stem cels into myocardial celsin vitro, and differentiated cels expressed desmin, α-actin, cTnT, cTnI and P38MAPK. The optimal induced concentration of dimethyl sulfoxide was 1.0%. Immunofluorescence double staining and electron microscope results further confirmed that dimethyl sulfoxide could induce the myocardial differentiation of bone marrow mesenchymal stem cels. Cite this article:Sun LY. Dimethyl sulfoxide induces differentiation of bone marrow mesenchymal stem cels into myocardial cels. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):26-30.
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BACKGROUND:Stem cels are induced to differentiate into endothelial-like cels that can be used for the treatment of diabetic lower extremity vascular disease. However, it is unclear whether these endothelial-like cels can completely replace endothelial cels to improve vascular disease and what are the differences between endothelial-like cels and endothelial cels. OBJECTIVE:To explore the differences and similarities between endothelial-like cels and human umbilical vein endothelial cels in the aspects of morphology, function, and viability. METHODS:Umbilical cord mesenchymal stem cels and umbilical vein endothelial cels were isolated, cultured and identified using flow cytometry and immunohistochemical method. Isolated umbilical cord mesenchymal stem cels were induced in DMEM-LG/F12 containing 10 μg/L vascular endothelial growth factor, 10 μg/L basic fibroblast growth factor and 2% fetal bovine serum to differentiate into endothelial-like cels folowed by immunohistochemical identification. To compare endothelial-like cels with human umbilical vein endothelial cels, cel migration detection, active substance measurement and three-dimensional angiogenesis test were performed. RESULTS AND CONCLUSION:Isolated umbilical cord mesenchymal stem cels strongly expressed the surface markers of mesenchymal stem cels, and human umbilical vein endothelial cels strongly expressed CD31 and VWF. After induction, the umbilical cord mesenchymal stem cels were identified to highly express CD31 and VWF. Through cel migration, active substance and three-dimensional angiogenesis tests, endothelial-like cels were similar to endothelial cels in the function and activity, and superior to endothelial cels. Cite this article:Hao XJ, Hao HY, Zhu MJ, Yuan Z, Li WW, Chen J, Zhu LY. Endothelial-like cels versus human umbilical vein endothelial cels. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):83-88.
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BACKGROUND:Currently, there is a lack of efficient, non-invasive way to transplant stem cels to the target organ or tissue. Exploring a way to guide targeting transplantation of stem cels and to improve the efficiency of stem cel homing is now one of focuses in the field of stem cels research. OBJECTIVE: To establish a simple and feasible method to chemicaly modify the cel surface using biotin-streptavidin reaction system, and to evaluate the efficiency of this method to label bone marrow mesenchymal stem cels (BMSCs) and its effects on cel biological functions. METHODS: Passage 3 BMSCs were obtained by whole bone marrow culture method and verified by flow cytometry. Biotin, streptavidin, sulfonated biotin-N-hydroxy-succinimide were used to equip the adhesion molecule ligand, sialyated LewisX (SLeX), to the BMSCs surface. The labeling rate of BMSCs was assessed using fluorescence microscope, the vitality of BMSCs was evaluated by trypan blue staining, and the proliferation of BMSCs was evaluated by cel counting kit-8 assay. Adipogenic and osteogenic inductions were used to evaluate the effect of the method on the multi-differentiation function of BMSCs. RESULTS AND CONCLUSION: After culture for 2 weeks, passage 3 BMSCs were obtained and confirmed by expressing CD90, CD29 and lack of CD34, CD45. Biotin, streptavidin, sulfonated biotin-N-hydroxy-succinimide were successfuly used to equip sialyated LewisX (SLeX) to the BMSCs surface and had minor effects on the vitality, proliferation, and differentiation of BMSCs. This method was simple for surface modification and had a high modification rate of 88%. The homing of BMSCs modified by this method to the target organ or tissue could be greatly enhanced. Therefore, this method potentialy could have extensive and important applications.
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Abstract BACKGROUND: Alveolar bone deficiency wil not meet aesthetic and functional requirements for dental implants. OBJECTIVE:To observe the repair effect of passage 3 autologous bone marrow mesenchymal stem cels (BMSCs) and platelet-rich fibrin (PRF) on alveolar bone defects in rabbits. METHODS:Twenty-seven New Zealand rabbits were randomly divided into BMSCs/PRF group, PRF group and model group (n=9 per group). The left mandible incisors were extracted in al the rabbits under general anesthesia. BMSCs/PRF group was immediately implanted BMSCs/PRF composite into the alveolar socket, PRF group only implanted PRF, and model group implanted nothing. RESULTS AND CONCLUSION: In the model group, the alveolar crest and alveolar mucosa become sunken notably and narrowed. In the BMSCs/PRF and PRF groups, the thickness of alveolar bone wal, alveolar bone width, alveolar bone height difference, and bone mineral density were al increased, especialy in the former group. In addition, the trabecular arrangement was better in the BMSCs/PRF groups than the model and PRF group. Our findings indicate that alveolar socket filing with composite of BMSCs and PRF can achieve preservation of alveolar bone width and height after tooth extraction in rabbits.
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BACKGROUND:Ankylosing spondylitis is an autoimmune disease at high inflammatory state, and its pathogenesis is stil unclear. Besides, there is a lack of entirely satisfactory curative strategies. OBJECTIVE: To explore the immunoregulation capability of bone marrow mesenchymal stem cels from ankylosing spondylitis patients on macrophages and the potential therapeutic use of bone marrow mesenchymal stem cels from healthy donors on ankylosing spondylitis. METHODS: Bone marrow mesenchymal stem cels were extracted from 21 healthy donors and 25 ankylosing spondylitis patients respectively, and passage 4 cels were used in subsequent experiments. A human monocytic cel line was induced to differentiate into macrophages. The phenotypic markers of bone marrow mesenchymal stem cels and macrophages were detected by flow cytometry. Expressions of tumor necrosis factor-α and tumor necrosis factor-α-stimulated gene 6 (TSG-6) proteins in the supernatant of co-culture system were detected by ELISA. Quantitative real-time PCR was applied to detect the mRNA level of cytokines secreted by bone marrow mesenchymal stem cels and macrophages. RESULTS AND CONCLUSION:The typical mesenchymal stem cel surface markers were expressed in both bone marrow mesenchymal stem cels from healthy donors and patients with ankylosing spondylitis, and CD68 was detected positively in induced macrophages. The protein and mRNA levels of tumor necrosis factor-α secreted by macrophages co-cultured with bone marrow mesenchymal stem cels from patients with ankylosing spondylitis were obviously higher than those from healthy donors (P < 0.05). TSG-6 secreted by bone marrow mesenchymal stem cels from patients with ankylosing spondylitis was lower than that by bone marrow mesenchymal stem cels from healthy donors in both RNA transcriptional and protein levels (P < 0.05). Our study demonstrates that bone marrow mesenchymal stem cels from patients with ankylosing spondylitis shows abnormal immunoregulatory function on inhibiting the tumor necrosis factor-α secretion from macrophages, which reveals a mechanism of immune disorder in ankylosing spondylitis. The therapeutic mechanism of bone marrow mesenchymal stem cels from healthy donors may work by secreting enough TSG-6 to inhibit the activation of macrophages in patients with ankylosing spondylitis, and thereby to decrease the secretion of tumor necrosis factor-α. Cite this article:Sun SH, Wang P, Su CY, Xie ZY, Li YX, Li D, Wang S, Su HJ, Wu XH, Deng W, Wu YF, Shen HY. Bone marrow mesenchymal stem cels derived from patients with ankylosing spondylitis show abnormal immunoregulation capability on macrophages. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):13-19.
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BACKGROUND:It wil provide a new insight into the future application of bone marrow mesenchymal stem cels in the treatment of spinal cord injury and tissue engineering by studying the effect of activation of Wnt signaling pathway in the neuronal differentiation of bone marrow mesenchymal stem cels. OBJECTIVE: To detect the expression of related genes by gene chip technology during the neuronal differentiation of bone marrow mesenchymal stem cels. METHODS:Human bone marrow mesenchymal stem cels were isolated and purified, and passage 5 cels were obtained. GatewayTM technology was used to build lentiviral vectors that was used to transfect Wnt-1 into human bone marrow mesenchymal stem cels. Control, non-transduction and transduction groups were set in this study. Human bone marrow mesenchymal stem cels were then induced to differentiate into neurons. Cel morphology was observed under inverted phase contrast microscope. Gene chip was used to detect the regulation changes and the differential expression of related genes in the Wnt signaling pathway. RESULTS AND CONCLUSION: Under the scanning electron microscope, the transfected cels were found to have the similar morphology of neuron-like cels. Analysis by the gene chip hybridization technique showed that 3 287 genes were up-regulated and 4 215 genes were down-regulated in the signal pathway. In the Wnt signaling pathway, genes related to the nervous system development and differentiation were up- or down-regulated. It is verified that the Wnt signal pathway is activated via Wnt-1 transduction, and the downstream genes appear to have genetic transcription so as to promote the neuronal differentiation of human bone marrow mesenchymal stem cels.
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BACKGROUND:We attempt to explore a low-cost, simple and effective way to cryopreserve bone marrow mesenchymal stem cels at-80℃. OBJECTIVE:To screen the optimal cryopreservation fluid for bone marrow mesenchymal stem cels and to verify the biological features of bone marrow mesenchymal stem cels after long-term cryopreservation. METHODS: Bone marrow mesenchymal stem cels were cultured using adherent method and the biological features and purity of cels were detected using immunofluorescence method. Bone marrow mesenchymal stem cels were cryopreserved in the cryoprotectant medium containing low-sugar DMEM, fetal bovine serum and dimethyl sulfoxide at different proportions at-80℃ for a short term. Then, the optimal cryoprotectant was selected to storage the bone marrow mesenchymal stem cels. After 1, 3, 6 months of cryopreservation, the cels were resuscitated, cultured and passaged. Passage cels were identified immunofluorescence method to determine the biological features of bone marrow mesenchymal stem cels cryopreserved at-80℃. RESULTS AND CONCLUSION:Cryoprotectant medium of 80% DMEM+10% fetal bovine serum+10% dimethyl sulfoxide was suitable for cryopreserving MSCs at -80℃, and resuscitated cels were able to proliferate in vitro, and passage normaly, indicating the cryopreserved bone marrow mesenchymal stem cels stil maintain the original biological activity.
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BACKGROUND:Recently, the role of mesenchymal stem cels in aplastic anemia has been widely explored. However, its underlying mechanism remains unclearly. OBJECTIVE: To study the effect of umbilical cord blood and bone marrow mesenchymal stem cels on hematopoietic support and secretory function of T lymphocytes in patients with aplastic anemia. METHODS: Cord blood and bone marrow samples from 48 cases of aplastic anemia and 48 healthy lying-in women to isolate mesenchymal stem cels using flow cytometry. Mesenchymal stem cels from the cord blood and bone marrow were respectively co-cultured with cord blood mononuclear cels to count burst forming units-erythroid and colony forming units-granulocyte/macrophage. Mesenchymal stem cels were co-cultured with T lymphocytes from aplastic anemia patients undergoing phytohemagglutinin stimulation, and ELISA was used to detect interleukin-2, interleukin-4 and interferon-γ levels secreted from T lymphocytes. RESULTS AND CONCLUSION:The number of burst forming units-erythroid and colony forming units-granulocyte/macrophage significantly increased in normal bone marrow or umbilical cord blood mesenchymal stem cels co-cultured with cord blood mononuclear cels (P < 0.05), but reduced remarkably in umbilical cord blood mesenchymal stem cels from aplastic anemia patients co-cultured with cord blood mononuclear cels (P < 0.05). Levels of interleukin-2, interleukin-4 and interferon-γ from T lymphocytes were inhibited significantly after co-culture with normal bone marrow mesenchymal stem cels compared with phytohemagglutinin-induced T lymphocytes (P < 0.05). There was a similar inhibitory effect after co-culture with normal umbilical cord blood mesenchymal stem cels. There was a significantly reduction in the capacity of inhibiting interleukin-2, interleukin-4 and interferon-γ levels from T lymphocytes after co-culture with bone marrow mesenchymal stem cels from aplastic anemia patients (P < 0.05). Aplastic anemia patients show some functional defects in their bone marrow mesenchymal stem cels that have a weaker inhibitory role than normal bone marrow or umbilical cord blood mesenchymal stem cels in the hematopoietic support and secretory function of T lymphocytes. These findings indicate that mesenchymal stem cels from aplastic anemia patients can influence the pathological progress through weakening hematopoietic support and secretory function of T lymphocytes. Cite this article:Li GC, Song YP, Zhang YJ, Li G, Wang H, Xie J.Effects of mesenchymal stem cels on hematopoietic support and secretory function of T lymphocytes in patients with aplastic anemia. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):107-112.
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BACKGROUND:In recent years, in-depth studies that single Chinese herbs or extracts, compound traditional Chinese medicine and medicated serum are used to regulate the directional differentiation of bone marrow mesenchymal stem cels into myofibroblasts, chondrocytes, osteoblasts, myocardial cels and nerve cels, which have become a highlight in the tissue engineering research. OBJECTIVE:To review the latest progress in the directional differentiation of bone marrow mesenchymal stem cels induced by Chinese herbs or their extracts. METHODS:The first author searched the CNKI, Wanfang and PubMed databases using the keywords of “Chinese herb, directional differentiation, mesenchymal stem cels” in Chinese and English, respectively, to retrieve relevant articles published from January 2010 to January 2016. Repetitive articles or those with no originality were eliminated. Totaly 99 articles were searched initialy, and then 43 articles were included in result analysis. RESULTS AND CONCLUSION:As the strongest seed cels in the bone differentiation system, bone marrow mesenchymal stem cels have a wide range of directional differentiation potential, and highlight the important value in combination with Chinese herbs for clinical treatment of various refractory diseases, especialy for treatment of metabolic bone diseases, bone defects, nonunion and delayed union, which is not only conducive to in-depth, multi-angle studies on effects and mechanisms of Chinese herbs, but also to clinical treatment of various refractory diseases using bone marrow mesenchymal stem cels. Cite this article:Li N, Li YF, Xie XW, Song M, Xu SH, Li DP.Directional differentiation of bone marrow mesenchymal stem cel induced by traditional Chinese Medicine. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):135-139.
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BACKGROUND:Bone marrow mesenchymal stem cels (BMSCs) can promote nerve regeneration, but there are no better results because of the limitations of treatment methods. BMSC transplantation alone is not enough to achieve desired therapeutic effects. OBJECTIVE:To investigate the effect of fibroblast growth factor (FGF)-modified BMSC transplantation on functional recovery and expression of glial fibrilary acidic protein after traumatic brain injury. METHODS:Animal models of traumatic brain injury were established in Sprague-Dawley rats using hydraulic shock method, and then randomized into control group (traumatic brain injury group), BMSC group and FGF-BMSC group (FGF-modified BMSC group). After isolation and culture, BMSCs were modified by adenovirus vector-mediated FGF gene. Western blot assay was used to detect transfection efficiency and glial fibrilary acidic protein expression; immunohistochemical detection was used to detect distribution and number of BrdU positive cels in the brain; Longa score was used to evaluate the neurologic function of rats at 1, 3 days, 1, 2 weeks after transplantation; TUNEL assay was used to detect cel apoptosis in the brain. RESULTS AND CONCLUSION:Western blot results showed that FGF gene was successfuly transferred to the adenovirus vector, and capable of expressing in BMSCs; moreover, the glial fibrilary acidic protein expression of FGF-BMSC group was significantly higher than that in the other two groups (P < 0.05). The number of BrdU positive cels in the brain was significantly higher in the FGF-BMSC group than the other two groups (P < 0.05). Two weeks after transplantation, the Longa scores in the FGF-BMSC group were significantly lower than those in the other two groups (P < 0.05). TUNEL results showed that the number of apoptotic cels in the FGF-BMSC group was significantly lower than that in the other two groups (P < 0.05). These findings indicate that FGF-modified BMSCs transplantation is able to improve neurological damage after traumatic brain injury and promote neurological recovery, which is better than BMSC transplantation alone.
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BACKGROUND:Mesenchymal stem cels have the specific chemotaxis to the inflammation and tumor tissue, but the effect of mesenchymal stem cels on the growth of cervical cancer cels becomes an urgent problem. OBJECTIVE:To investigate the effect of human umbilical cord mesenchymal stem cels on the proliferation of cervical cancer Hela cels. METHODS:Hela cels were co-cultured with human umbilical cord mesenchymal stem cels at different number or its conditioned medium at different concentrations for 3 days. Then, cel counting kit-8 was used to detect the proliferation of Hela cels. RESULTS AND CONCLUSION:When Hela cels were co-cultured with human umbilical cord mesenchymal stem cels at ratios of 1:3, 1:2, 1:1, 2:1, 4:1, 8:1, the relative proliferation inhibition rates were 67.12%, 47.18%, 31.15%, 27.61%, 15.55% and 15.95%, respectively. When the Hela cels were co-cultured with human umbilical cord mesenchymal stem cel conditioned medium at 5, 10, 20, 40, 60, 100 mg/L, the relative proliferation inhibition rates were 0.61%, 40.1%, 63.47%, 80.61%, 93.56%, 90.65%, respectively. These findings indicate that the proliferation of Hela cels can be inhibited by co-culture with human umbilical cord mesenchymal stem cels at a certain concentration-dependent manner.
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BACKGROUND:With the continuous development and progress of medical model, the treatment and rehabilitation recovery from peripheral nerve defects needs higher requirements; therefore, stem cel culture-based nerve tissue engineering technology provides a new strategy for nerve defect repair. OBJECTIVE:To explore the feasibility of combining bone marrow mesenchymal stem cels and new nerve conduits to repair peripheral nerve defects. METHODS:Fifty New Zealand white rabbits, clean grade, were randomly divided into experimental group and control group, with 25 rabbits in each group. Animal models of peripheral nerve defects were made about 15 mm distant to the middle segment of the radius of the rabbit foreleg. At 1 week after modeling, bone marrow mesenchymal stem cels/new nerve conduit composite material was implanted into the defect in the experimental group; and in the control group, only bone marrow mesenchymal stem cels were transplantation. At 4 weeks after transplantation, a 5-mm nerve fiber was taken from the defect site, stained with hematoxylin-eosin and observed under scanning electron microscope. Density and diameter of regenerated nerve fibers were compared between the two groups. RESULTS AND CONCLUSION:Compared with the control group, the density of nerve fibers was significantly higher in the experimental group, but the diameter of nerve fibers was significantly lower (P < 0.05). There were more cels with good growth, large size and many processes on the surface of regenerated nerve tissues in the experimental group than the control group. Moreover, in the experimental group, interconnected cels were woven into a mesh and the axons were longer and thicker, both of which were the performance of typical neuron-like cels. Taken together, the bone marrow mesenchymal stem cels/new nerve conduit composite material can be used for peripheral nerve repair and present exact achievements.
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BACKGROUND:Active components of Astragalus have an antioxidant effect, which is considered to result in the neuron-like differentiation of bone marrow mesenchymal stem cels. OBJECTIVE:To investigate the effect of astragalus polysaccharides combined with basic fibroblast growth factor to induce the differentiation of bone marrow mesenchymal stem cels into nerve cels. METHODS:After 24 hours of pretreatment with basic fibroblast growth factor, passage 3 human bone marrow mesenchymal stem cels were cultured with astragalus polysaccharides for 1-3 days (combined group). Blank control group and basic fibroblast growth factor group were set up. Expression of neuron-specific enolase and nestin was detected using western blot or immunocytochemical staining. RESULTS AND CONCLUSION:The expression of neuron-specific enolase was higher in the combined group than the basic fibroblast growth factor group (P < 0.05). Expression of nestin was found in both basic fibroblast growth factor group and combined group, but the gray value was higher in the combined group than the basic fibroblast growth factor group (P < 0.05). These findings indicate that astragalus polysaccharides combined with basic fibroblast growth factor is better to induce the neuronal differentiation of bone marrow mesenchymal stem cels.
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BACKGROUND:Cobalt chloride (CoCl2) may promote the proliferation of human umbilical cord-derived mesenchymal stem cels (hUC-MSCs) in a time- and concentration-dependent manner, and meanwhile, CoCl2 can regulate the expression of genes and proteins in hUC-MSCs. OBJECTIVE:To explore the effects of CoCl2 induced-hypoxia on the proliferation of hUC-MSCs and gene and protein expressions in hUC-MSCs, thereby establishing an effective method for MSCs culture and amplificationin vitro. METHODS: hUC-MSCs were extracted using tissue explant method. Under hypoxia conditions induced by CoCl2 (0, 100, 150, 200, 250 μmol/L) for different periods (0, 1, 2, 3, 4 days), flow cytometry was used to identify cel surface-associated antigens; cel counting kit-8 was used to detect cel proliferation; RT-PCR was used to determine levels of hypoxia inducible factor-1α, inducible nitric oxide synthase, stromal cel-derived factor-1, interleukin-6, transforming growth factor-β mRNA; western blot assay was used to detect protein expression of hypoxia inducible factor-1α. RESULTS AND CONCLUSION:The cels were positive for CD29, CD73, CD90, CD105, while negative for CD31, CD14, CD34, CD45, CD11b, HLA-DR. Moreover, the antigen expression was not affected by CoCl2 induced-hypoxia. CoCl2 induced-chemical hypoxia could promote the proliferation of hUC-MSCs in a time- and concentration-dependent manner. RT-PCR results showed thatunder hypoxia, hypoxia inducible factor 1α, inducible nitric oxide synthase and stromal cel-derived factor-1 mRNA expressions were significantly up-regulated, but interleukin-6 and transforming growth factor-β mRNA expressions were down-regulated significantly (P < 0.05). Additionaly, the protein expression of hypoxia inducible factor 1α was increased under hypoxia conditions. These findings indicate that CoCl2 induced-hypoxia environment may promote the proliferation of hUC-MSCs and the optimal concentration of CoCl2 is 200μmol/L. However, a higher concentration of CoCl2 (≥ 250μmol/L) inhibits the proliferation of hUC-MSCs, and the mechanism may be related to the increase of hypoxia inducible factor-1α at protein and mRNA levels.
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BACKGROUND:Conditioned medium from mesenchymal stem cels (MSC-CM) that contains abundant MSCs paracrine substances may represent a promising alternative to MSCs transplantation. However, normal MSC-CM with insufficient paracrine ability is not effective for tissue damage repair. OBJECTIVE:To investigate the effects of MSC-CM with (MSC-CMHyp) and without hypoxic activation (MSC-CMNor) on the proliferation and apoptosis of radiation-induced injured intestinal epithelial cels (IEC-6) and to further discuss the paracrine mechanisms. METHODS: IEC-6 cels were exposed to 10 Gy irradiation and cultured in MSC-CMHyp, MSC-CMNor, and DMEM-F12 medium, respectively. RESULTS AND CONCLUSION: Findings from trypan blue staining, flow cytometry and western blot assay showed that, compared with the DMEM-F12 medium group, treatment with MSC-CMHyp significantly enhanced IEC-6 viability proliferation after radiation-induced injury, as wel as significantly decreased cel apoptosis and expression of Caspases-3/8 (P 0.05). On the other hand, the increased levels of vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-10 were detected in the MSC-CMHyp group compared to the MSC-CMNor group (P < 0.05). These results suggest that the MSC-CMHyp improves the viability and proliferative capacity of IEC-6 cels after radiation-induced injuryvia up-regulating secretion of cytokines and down-regulating apoptotic signaling.
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BACKGROUND:Articular cartilage is a highly diferentiated tissue, which is very limited in its ability to repair after injury. Stem cel therapy for cartilage repair has completely solved this problem. OBJECTIVE: To investigate the mechanism that diferent growth factors induce the diferentiation of bone marrow mesenchymal stem cels into chondrocytes. METHODS:Passage 5 rat bone marrow mesenchymal stem cels were induced by diferent growth factors and their combinations, including transforming growth factor beta 1 (TGF-β1) group, TGF-β1+insulin growth factor-1 (IGF-1) group, and bone morphogenetic protein 2 (BMP-2)+IGF-1 group, TGF-β1+BMP-2 group, TGF-β1+IGF-1+ BMP-2 group, and blank control group. At 21 days of induction, cels were stained with alcian blue and alizarin red; RT-PCR was employed to detect colagen I mRNA expression. RESULTS AND CONCLUSION:Alcian blue staining showed metachromasia in the cytoplasm and mesenchyma, and proteoglycan was expressed green; alizarin red staining showed no orange calcium nodules. The bone marrow mesenchymal stem cels were preliminarily deduced to diferentiate into chondrocytes, but could not express the cel phenotypes of bone cels. In the blank control group, the expression of colagen I mRNA was negative. Compared with the TGF-β1 group, the mRNA expression of colagen I was lower in the BMP-2+IGF-1 group, but higher in the TGF-β1+BMP-2 group and TGF-β1+IGF-1+BMP-2 group (P < 0.05). Moreover, the expression of colagen I mRNA was highest in the TGF-β1+IGF-1+BMP-2 group (P < 0.05). These findings indicate that TGF-β1 alone is able to induce the chondrogenic diferentiation of bone marrow mesenchymal stem cels, and the combination of TGF-β1, IGF-1 and BMP-2 can play the biggest role to induce the chondrogenic diferentiation of bone marrow mesenchymal stem cels.
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BACKGROUND:Fetal bovine serum as nutritional support is often used in the traditional cel culture. Consequently, a host of potential problems such as the spread of disease and immunological reactions exist. To find a suitable fetal bovine serum substitute and to establish a culture system of human bone marrow stromal stem cels in vitro which has been standardized, safe and efficient has just started. OBJECTIVE:To investigate the effects of different serums on proliferation of bone marrow stromal stem celsin vitro. METHODS:Bone marrow stromal stem cels were obtained from adult bone marrow, which were cultured in DMEM containing 10% AB serum, 10% autologous serum, or 10% fetal bovine serum. Cels at passage 3 were used in this study. RESULTS AND CONCLUSION:The cel confluence in the AB serum group was earlier than that in the fetal bovine serum group and autologous serum group. Human bone marrow stromal stem cels maintained the phenotypes of bone marrow stem cels in three serums detected by flow cytometry. AB serum group showed the highest fluorescence intensity and the most efficiency of cel proliferation which examined by the AlamarBlue assay. Apoptosis rate was < 5% in al the three groups, and cels grew wel in these serums. Alkaline phosphatase, calcium nodules and oil red O staining showed that the cels maintained the osteogenesis and adipogenesis capacity in the three groups. AB serum was found to have a better effect on proliferation capability of cels than fetal bovine serum and autologous serum. Taken together, AB serum is expected to be a substitute of fetal bovine serum to build anin vitro culture system of adult bone marrow stromal stem cels that accord with the clinical requirements of bone tissue engineering.
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BACKGROUND:Trauma easily leads to the emergence of articular cartilage defects, which is a difficult problem in the orthopedics field. Tissue engineering technology provides a new method for cartilage repair. OBJECTIVE:To explore the feasibility of combining chitosan and bone marrow mesenchymal stem cels to repair injured articular cartilage in rabbits. METHODS: Cultured rabbit bone marrow mesenchymal stem cels were seeded onto chitosan scaffold, and then the composite material was implanted into the defect as experimental group. Rabbits with no treatment served as control group. Gross observation and toluidine blue staining were carried out at 6 and 12 weeks after operation. RESULTS AND CONCLUSION:At 6 weeks after operation, the control group had only fibrous tissue hyperplasia, and in the experimental group, cartilage-like tissues were generated at the defect site. At 12 weeks after operation, a smal amount of hyaline cartilage-like tissues were observed in the control group, and the defects in the experimental group were covered with smooth and hyaline cartilage tissues. After 12 weeks, the toluidine blue staining was light in the control group with a smal amount of cartilage tissues; in the experimental group, the toluidine blue staining was remarkable, and the defects were completely covered with hyaline cartilage tissues, and cartilage cels were increased in number. The findings indicate that chitosan-bone marrow mesenchymal stem cels composite material is better to induce cartilage tissue formation and promote cartilage defect repair.
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BACKGROUND:There is no clear understanding about the effect of let-7f and interleukin-6 (IL-6) on the proliferation of bone marrow mesenchymal stem cels and their relationship. OBJECTIVE: To explore the effects of expression levels of let-7f and IL-6 on the proliferation of bone marrow mesenchymal stem cels and their relationship. METHODS:(1) LV-rno-let-7f-up and LV-rno-let-7f-down were constructed and transfected into bone marrow mesenchymal stem cels of Sprague-Dawley rats, respectively. Then, there were four groups in the study: transfection upregulation group transfected with LV-rno-let-7f-up), transfection inhibition group (transfected with LV-rno-let-7f-down), negative control group (transfected with FU-RNAi-NC-LV), and untransfected group. The expression level of let-7f in each group was detected by qRT-PCR. The proliferation ability of cels and expression levels of IL-6 when let-7f expression was at different levels were detected by MTT, flow cytometry and ELISA. The expression of Cyclin D1 at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. (2) To predict the potential target gene of let-7f, the wild-type/mutant IL-6 3’UTR reporter gene vectors were constructed, and cotransfected with let-7f/let-7f inhibitor respectively into the 293T cels to measure the luciferase. RESULTS AND CONCLUSION: Compared with the negative control group, the proliferative and cloning capacities of cels in the transfection upregulation group were higher; the number of cels was significantly decreased at G1 stage and increased at S stage, and the apoptotic cels were reduced in number (P 0.05). Luciferase activity of cels transfected with wide-type IL-6 3’UTR and let-7f was significantly reduced (P < 0.05). These findings indicate that up-regulation of let-7f can promote the proliferative and cloning capacities of bone marrow mesenchymal stem cels and reduce cel apoptosis, but downrelation of let-7f exhibits an inhibitory effect. Overexpression of IL-6 can suppress the proliferation of bone marrow mesenchymal stem cels, which is considered to be a target gene of let-7f, and let-7f may suppress the expression of IL-6 to promote the cel proliferation.