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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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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: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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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: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: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: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: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: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: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:At present, a lot of research about culture methods for umbilical cord mesenchymal stem cels, but not for the waste of primary system. OBJECTIVE:To explore the best culture method of human umbilical cord mesenchymal stem celsin vitro. METHODS:Human umbilical cord mesenchymal stem cels were prepared by tissue explants method, recorded as initial culture group. The centrifugal fluid and tissue of the primary culture flask were centrifuged and divided into three groups for secondary culture: tissue group, mixed group and pure liquid group. Cel morphology, time for cel acquisition, and yield of primary cels in the four groups were observed; the cel growth curve was analyzed by MTT assay; and cel cycle and phenotype were detected by flow cytometry. RESULTS AND CONCLUSION: The average time for cel acquisition in the initial culture group, tissue group, mixed group and pure liquid group were (15.00±0.45), (7.0±0.3), (8.00±0.25) and (8.00±0.25) days, respectively. The number of cels at first generation was (4.0±0.5)×105, (9.0±0.55)×105, (15.0±0.2)×105 and (7.0±0.33)×105 markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cels can be obtained rapidly and largely through the secondary culture to the primary culture system. T75 culture bottle, respectively. Under the inverted microscope, cels in the four groups were fusiform-like adherent cels, which were in paralel or circinate arrangement. Growth curve, proliferative activity, surface markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cells can be obtained rapidly and largely through the secondary culture to the primary culture system.
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BACKGROUND:The metastatic potential of hepatocelular carcinoma cels is key factor influencing patient’s prognosis. To observe the effect of human bone marrow mesenchymal stem cels on metastasis of hepatocelular carcinoma is of great significance for improving the lifetime of hepatocelular carcinoma patients. OBJECTIVE:To explore the biological effect of human bone marrow mesenchymal stem cels on hepatocelular carcinoma cels with different metastatic potentials. METHODS:Human bone marrow mesenchymal stem cels and hepatocelular carcinoma cel suspension with high and low metastatic potentials were respectively injected into the Transwel chamber, and after 36 hours of co-culture, ELISA method was used to detect the absorbance value as wel as cel counting method was used to observe the changes in the invasion ability of hepatocelular carcinoma cels. The effects of human bone marrow mesenchymal stem cels on the proliferation of hepatocelular carcinoma cel suspension with high and low metastatic potentials were determined using cel counting kit-8. PCR method was adopted to measure the expression of osteopontin, bone specific sialoproteins, integration (alpha V), transforming growth factor beta 1 and programmed cel death protein 5. RESULTS AND CONCLUSION:(1) The number of migrated hepatocelular carcinoma cels was significantly lower in the co-culture group than the single culture group, and based on the semi-quantitative detection of invasion ability, the absorbance value of the co-culture group was significantly lower than that in the single culture group (P 0.05). In the co-culture group with low metastatic potential, the expression of osteopontin, bone specific sialoproteins, and integration (alpha V) were declined remarkably (P 0.05). However, in the co-culture group with low metastatic potential, the expression of transforming growth factor beta 1 and programmed cel death protein 5 was both increased dramaticaly (P < 0.05). These findings suggest that the human bone marrow mesenchymal stem cels reduce the invasion ability of hepatocelular carcinoma cels, and enhance their ability of proliferation.
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BACKGROUND:Previous studies have found that the basement membrane matrix can induced the chondrogenic differentiation of bone marrow mesenchymal stem cels, but there is a wide gap between its mechanical properties and practical application, and further research is needed. OBJECTIVE:To prepare a suitable matrigel/chitosan scaffold that has appropriate mechanical properties and remarkable bioactivity for cartilage repair. METHODS: We selected genipin as cross-linking agent, and mixed Matrigel with cross-linked chitosan at different ratios (2:1, 1:1, 1:3). Then rat bone marrow mesenchymal stem cels were seeded on different scaffolds and cultured for 14 days. The mechanical properties of materials were measured by DMA. Cel counting kit-8, FDA staining, ELISA kits and Alcian blue staining were used to measure the bioactivity of materials. RESULTS AND CONCLUSION:The storage modulus of scaffolds was raised from 0.48 kPa to 1.78 kPa with increase ratio of chitosan. Cels spread wel in the early period on al scaffolds, and then the cels on the chitosan scaffold showed reduced chondrogenic activity, but cels on the scaffolds with matrigel could maintain chondrogenic differentiation. The matrigel/chitosan scaffold at a ratio of 1:1 had appropriate mechanical properties and higher levels of colagen II and colagen X at 14 days. The prepared matrigel/chitosan scaffold with decent mechanical performance can promote the differentiation of bone marrow mesenchymal stem cels into chondrogenic lineages, which can be used in cartilage tissue engineering.
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BACKGROUND:Bone marrow mesenchymal stem cels are crucial for bone and cartilage development and regeneration at a celular level. Insufficient quantity and functional impairment of bone marrow mesenchymal stem cels is widely considered to be one of osteoarthritis causes. OBJECTIVE: To explore the relationship between the functional status of bone marrow mesenchymal stem cels and disease progression in osteoarthritis patients.METHODS: Thirty patients with osteoarthritis were enroled from July 2013 to October 2014, and divided into control, mild osteoarthritis, and severe osteoarthritis groups, with 10 cases in each group. 5 mL bone marrow from the femur or tibia was extracted from each patient to isolate and culture bone marrow mesenchymal stem cels. Proliferation ability of cels at passage 3 was detected using cel counting kit-8; toluidine blue staining was performed at 14 days after chondrogenic induction; real-time PCR was used to detect the mRNA expression of Aggrecan and Col2A1 in the control group after chondrogenic induction. RESULTS AND CONCLUSION:Afterin vitro culture, bone marrow mesenchymal stem cels grew adherently in polygonal and fusiform shape with multiple processes at uniform size. The cytoplasm contained larger particles and the nuclei were ovoid. Most of cels were in cel division phase. The proliferation ability was strongest in the control group and weakest in the severe osteoarthritis group. Cels from the three groups were al at plateau phase after 1 week culture. At 14 days after chondrogenic induction, the cels were polygonal and quasi-circular, and purple metachromatic granules distributed outside of the cytoplasm. The expression of Aggrecan and Col2A1 in the control group displayed an overexpression trend. These findings indicate that the functional status of bone marrow mesenchymal stem cels from osteoarthritis patients is negatively correlated with the severity of disease, which can influence the disease progression in osteoarthritis patients.
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BACKGROUND:The use of mesenchymal stem cels in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cels are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cels. OBJECTIVE:To observe the chondrogenic effect of human bone marrow mesenchymal stem cels transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury. METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cels were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cel counting kit-8 was used to determine the cel proliferation, the differentiation of bone marrow mesenchymal stem cels towards chondrocytes was verified by type II colagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II colagen and aggrecan mRNA expression at 21 days of chondrogenic induction. RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cels. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II colagen were significantly increased in the miR-221-3p/222-3p inhibition group and cel proliferation was also inhibited significantly compared with non-transduced cels or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cels could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cels.
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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.
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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: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: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: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.