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目的:探讨炎症因子转化生长因子(transforming growth factor-α,TNF-α)对SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成脂分化的影响及相关的分子机制。方法:将生长良好的第3代(P3)SD大鼠BMSCs分为对照组(Con组)、成脂诱导组(Ad组)和10 ng/m L TNF-α干预成脂诱导组(Ad+TNF-α组),每组各接种5块6孔板,培养14 d后,收集各组细胞的RNA和蛋白质。于第14天时间点处进行油红O染色和异丙醇萃取,酶标仪490 nm处检测吸光度(absorbance,A)值,观察各组成脂分化的程度。q RT-PCR检测各组基因Wnt10b、前体脂肪细胞因子-1(preadipocyte factor-1,Pref-1)、CCAAT区增强子结合蛋白-α(CCAAT/enhancer-binding protein-α,C/EBP-α)、过氧化物酶体增殖物激活受体-γ(peroxisome proliferator-activated receptor-γ,PPAR-γ)、脂肪酸结合蛋白-4(fatty acid binding protein-4,FABP-4)的m RNA表达水平。Western blot检测第14天3组基因β联蛋白(β-catenin)、PPAR-γ、C/EBP-α、FABP-4的蛋白表达水平。结果:TNF-α可以明显抑制SD大鼠BMSCs的成脂分化。在Ad+TNF-α组,油红O染色明显低于Ad组,异丙醇萃取后A值检测也明显低于Ad组(0.360±0.035 vs.0.770±0.025,P=0.000)。q RT-PCR结果显示,在Ad+TNF-α组,Wnt10b、Pref-1的m RNA水平(17.050±2.706,4.135±0.280)明显高于Con组(P=0.019,P=0.003)和Ad组(0.575±0.065,0.514±0.060)(P=0.020,P=0.006),而C/EBP-α、PPAR-γ、FABP-4的m RNA水平(0.200±0.012,0.768±0.030,0.883±0.048)明显低于Ad组(2.965±0.455,2.330±0.211,3.847±0.171)(P=0.019,P=0.000,P=0.001)。Western blot结果显示,诱导14 d后,Ad+TNF-α组的C/EBP-α、PPAR-γ、FABP-4蛋白表达水平(0.586±0.013,0.356±0.008,0.118±0.002)明显低于Ad组(1.082±0.018,0.840±0.112,1.094±0.038)(均P=0.000)。在Ad+TNF-α组,磷酸化β-catenin(P-β-catenin)的蛋白水平(0.648±0.005)明显低于Ad组(3.376±0.211)(P=0.000)。结论:TNF-α抑制SD大鼠BMSCs成脂过程分化相的作用可能与Wnt/β-catenin信号通路活性的增加和Pref-1的高表达有关,由此BMSCs的成脂分化停留在了前体脂肪细胞阶段。
Objective: To investigate the effects of transforming growth factor-α (TNF-α) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in SD rats and its related molecular mechanisms. Methods: BMSCs of 3rd generation (P3) SD rats were divided into control group (Con group), adipogenic induction group (Ad group) and 10 ng / mL TNF-α intervention adipogenic induction group (Ad + TNF-α group). Each group was inoculated with five 6-well plates. After 14 days of culture, the RNA and protein of each group were collected. Oil red O staining and isopropanol extraction were performed at the 14th day. The absorbance (A) value was measured at 490 nm on the microplate reader to observe the degree of lipid differentiation. The expression of Wnt10b, preadipocyte factor-1 (Pref-1) and CCAAT / enhancer-binding protein-α (C / EBP- α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid binding protein-4 (FABP-4) Level. Western blot was used to detect the protein expression of β-catenin, PPAR-γ, C / EBP-α and FABP-4 on the 14th day. Results: TNF-α significantly inhibited the adipogenic differentiation of BMSCs in SD rats. In Ad + TNF-α group, oil red O staining was significantly lower than Ad group, A value was significantly lower than Ad group after isopropanol extraction (0.360 ± 0.035 vs.0.770 ± 0.025, P = 0.000). q RT-PCR results showed that the m RNA levels of Wnt10b and Pref-1 in Ad + TNF-α group (17.050 ± 2.706 and 4.135 ± 0.280) were significantly higher than those in Con group (P = 0.019, P = 0.003) (0.575 ± 0.065, 0.514 ± 0.060) (P = 0.020, P = 0.006), while the m RNA levels of C / EBP-α, PPAR-γand FABP- (2.965 ± 0.455,2.330 ± 0.211,3.847 ± 0.171) (P = 0.019, P = 0.000, P = 0.001). Western blot results showed that the expression levels of C / EBP-α, PPAR-γ and FABP-4 protein in Ad + TNF-α group were significantly lower than that in Ad-TNF-α group after 14 d of induction (0.586 ± 0.013,0.356 ± 0.008,0.118 ± 0.002) (1.082 ± 0.018, 0.840 ± 0.112, 1.094 ± 0.038) (all P = 0.000). The protein level of phosphorylated P-catenin (0.648 ± 0.005) in Ad + TNF-α group was significantly lower than that in Ad group (3.376 ± 0.211) (P = 0.000). CONCLUSION: The inhibitory effect of TNF-α on differentiation of BMSCs into adipocytes may be related to the increase of Wnt / β-catenin signaling pathway and the high expression of Pref-1, leading to adipogenic differentiation of BMSCs in the precursor Adipocyte stage.