TNF-α对SD大鼠骨髓间充质干细胞成脂分化的影响及其相关分子机制的探讨

发布时间：2018-04-03 04:33

  本文选题：骨髓间充质干细胞　切入点：成脂分化　出处：《重庆医科大学学报》2016年10期

【摘要】：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。
[Abstract]:Aim: to investigate the effect of transforming growth factor- 伪 (TNF- 伪) on lipid differentiation of bone marrow mesenchymal stem cells in SD rats and its molecular mechanism.Methods: the third generation of P3SD rats with good growth were divided into two groups: control group (Con group), adipogenic induction group (Ad group) and 10 ng/m L TNF- 伪 intervention group (Ad TNF- 伪 group). Each group was inoculated with 5 6-well plates and cultured for 14 days.The RNA and protein of each group were collected.Oil red O staining and isopropanol extraction were performed at the 14th day, and absorbency absorbency (A) was detected at 490 nm.瑙傚療鍚勭粍鎴愯剛鍒嗗寲鐨勭▼搴，

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