| Objective :To investigate the effect of TNF-α on adipogenesis of SD rat BMSCs and the related molecular mechanisms, which may provide a theoretical basis for clinical treatment and prevention of obesity-related metabolic diseases.Methods:(1)The whole bone marrow adherence method was used to isolate、purify and culture bone mesenchymal stem cells(BMSCs) from SD rats.(2)To identify the BMSCs, the well grown third passage(P3) cells were induced to osteogenesis and adipogenesis,and cell surface markers(CD45, CD11 b, CD44, CD90, CD34)were assessed by flow cytometry.(3) The MTT assay was used to evaluate the effect of TNF-α( 1、10、20、50、100ng/ml) on the growth of BMSCs at 24h、48h and 72 h. To explore the effect of TNF-α( 1、10、20、50、100ng/ml) on adipogenesis of SD rat BMSCs,cells were induced to differentiate into adipocytes and stained with Oil Red O, Optical density for Oil Red O was determined by using Isopropanol extraction and was measured in a plate reader at 520 nm.(4)The well grown third passage(P3) BMSCs were divided into three groups, i.e.Control, Ad and Ad+TNF-α groups, the RNA in three groups were collected in 3、7、14 days. The expressions of wnt 10 b, PPAR-γ, C/EBP-α, FABP-4 and Pref-1 at mRNA levels were analyzed by RT-PCR.(5)The protein of three groups were collceted in 14 days. The protein expression levels of β-catenin, C/EBP-ɑ, PPAR-γ, FABP-4 were detected by western blotting analysis.Results:(1)The cells that we isolated、purified and cultured from SD rat were spindle cell-based with abundant cytoplasm, large nucleia and prominent nucleoli, growing in a radial colony arrangement way. The cells were negative for CD45 、 CD11 b, positive for CD44、CD29、CD90. Following induction, cells can go through adipogenesis and osteogenesis.(2)The result of MTT demonstrated that TNF-α prevent the growth of BMSCs in a time dose dependent manner. From the culture of the 48 h, at the same time point, the cell proliferation rate was negatively correlated with the concentration of TNF-α; Under the same concentration of TNF-α, cells proliferation rate was negatively correlated with the incubation time. With the extension of the culture time, there was no statistically significant differences on the proliferation rate of BMSCs among higher concentrations(20-100ng/ml)of TNF-α(P> 0.05).(3) Optical density for Oil Red O determined as OD value showed that TNF-αcan inhibite the adipogenesis of BMSCs and the inhibition was positively correlated with the concentration of TNF-α.(4)RT-PCR showed that the mRNA expression levels of wnt10 b and Pref-1 in Ad+TNF-αgroup were significantly higher than in Ad group(P<0.05), and the mRNA expression levels of wnt10 b and Pref-1 in Ad+TNF-αgroup were positively correlated wtih the time of induction. While the mRNA expression levels of C/EBP-α,PPAR-γand FABP-4 were significantly lower in Ad+TNF-αgroup than in Ad group(P<0.05).(5)Western blot showed that after induction of 14 days,in Ad+TNF-αgroup, the protein expression levels of C/EBP-α, PPAR-γ, P-β-catenin and FABP-4 were lower than in Ad group(P<0.05).Conclusion:(1)The results of multi-directional differentiation and identification of cell surface markers show that the cells that we isolated、purified and cultured from SD rat are BMSCs.(2)TNF-αprevents the growth of BMSCs in a time dose dependent manner; TNF-α can inhibit the adipogenesis of SD rat BMSCs, this inhibition was positively correlated with the concentration of TNF-α.(3)TNF-α may by increasing the acticity of wnt / β-catenin signaling pathway inhibit the key transcription factors of adipogenesis-C/EBP-α 、 PPAR-γ, and finalily prevent the process of adipogenesis.The higher expression of Pref-1 in Ad+TNF-α group suggests that TNF-α may inhibit the terminal differentiation of the adipogenesis of SD rat BMSCs, while has no effect on determination phase. |
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