| BackgroundMesenchymal stem cells(MSCs)are a major member of multipotent stem cells family which can be isolated from bone marrow,fat,and some other tissues of fetal or adult.They have ability to self-renew and multipotential to differentiate into a variety of cell lineages including osteoblasts,adipocytes,chondrocytes or neurocytes etc.MSCs have important value in regenerative medicine and tissue repair damage.Cytokines,popular micro-molecule polypeptides which are secreted by immunocytes or non-immunocytes,mainly include interleukins,transforming growth factors and colony stimulating factor,interferons,tumor necrosis factor and so on.Moreover,cytokines have been identified to play a significant role in cell proliferation and differentiation.In this study,we aimed to investigate the effect of cytokines on the differentiation of mesenchymal stem cells and ascertain the underlying mechanisms.Objectives1 To explore the role of IL-6 in chondrocyte differentiation of bone marrow-derived mesenchymal stem cells in vitro.2 To determine the potential mechanism of IL-6 in chondrocyte differentiation of bone marrow-derived mesenchymal stem cells in vitro.3 To explore the role of TGF-?1 in adipogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.4 To ascertain the potential mechanism of TGF-?1 in adipogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.Methods: 1 We have set up the standard model for cartilage cell differentiation,and use only 6-10 generations of BMSC in mice as the research object.1.1 By observing the expression of IL-6 in the process of chondrocyte differentiation of BMSCs,we confirmed whether IL-6 influence the chondrocyte directional differentiation of BMSCs;1.2 The BMSCs were induced into chondrocyte in presence of recombinant IL-6,using Alcian blue staining analyse chondrogenic differentiation of BMSCs.Moreover,q PCR analysed the expression levels of cartilage differentiation related markers.Western blot was used to test the key chondrogenic differentiation transcription markers,Runx2 and Sox9.1.3 BMSCs were transitorily treated with the recombinant IL-6,then Western blot tested the levels of phosphorylation in the related signal pathways to detect which signal pathway can be activated.1.4 Specific inhibitors were used to interdict the key kinases of signal pathway which were discovered by the last step.Western blot was used to analyze the expression levels of Runx2 and Sox9 to clarify the molecular mechanism of IL-6 regulation of BMSCs differentiate into chondrocyte.2 We have set up the standard model for adipose cell differentiation,and use only 6-10 generations of BMSC in mice as the research object.2.1 By observing the expression of TGF-?1 in the process of adipocyte differentiation of BMSCs,we confirmed whether TGF-?1 influence the adipocyte directional differentiation of BMSCs;2.2 The BMSCs were induced into adipocyte in presence of recombinant TGF-?1,using Oil red O staining analysed adipocyte differentiation of BMSCs.Moreover,q PCR analysed the expression levels of adipocyte differentiation related markers.Western blot was used to detect the production levels of the adipocyte transcription markers,C/EBP? and the ?P2.2.3 BMSCs were transitorily treated with TGF-?1,then Western blot tested the levels of phosphorylation in the related signal pathways to detect which signal pathway can be activated.2.4 Specific inhibitors were used to interdict the key kinases of signal pathway which were discovered by the last step.Western blot were performed to analyze the production of C/EBP? and ?P2 to clarify the molecular mechanism of TGF-?1 regulation of BMSCs differentiate into cartilages.Results:1 This study demonstrated that IL-6 m RNA was significantly downregulated during the chondrogenic differentiation of BMSCs,and the levels of secreted IL-6 protein in medium were markedly decreased as well.Additionally,recombinant IL-6 inhibited chondrogenic differentiation of BMSCs,accompanying with the reduction of chondrocyte markers,Runx2 and Sox9.Furthermore,recombinant IL-6 induced the activation of MAPK/ERK,and the presence of ERK-specific inhibitor rescued the expression of the chondrocyte markers,including the Runx2 and Sox9.2 In the present study,we demonstrated that TGF-?1 gene was significantly down-regulated in adipogenesis of BMSCs in vitro.Moreover,recombinant TGF-?1 inhibited adipogenic differentiation of BMSCs in vitro,as evidenced by reduced induction of adipocyte markers,C/EBP? and ?P2.TGF-?1 not only activated Smad pathway,but also blunted the activation of PI3K/Akt signaling induced by insulin(key inducer of adipogenesis).In the presence of Smad-specific inhibitor,the inhibition of TGF-?1 on C/EBP? and ?P2 induction was abolished.Meanwhile,the inhibitory of TGF-?1 on PI3K/Akt signaling was also rescued.Conclusion:1 These results indicate that IL-6 can inhibit the chondrocyte differentiation of BMSCs via MAPK/ERK pathway activating.Our data also suggest that inflammatory factor IL-6 may disfavor the chondrocyte regeneration,providing some valuable references for studies of cartilage tissue engineering and therapy of cartilage diseases,such as osteoarthritis.2 The results suggest that TGF-?1 may regulate adipogenic differentiation of BMSCs by inhibiting PI3K/Akt activation via Smad pathway.Our data raise the possibility of using TGF-?1 intervention in the therapy of obesity-related diseases,such as type 2 diabetes and cardiovascular diseases. |
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