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  

Effect of TNF-α on adipogenesis of SD rat bone marrow mesenchymal stem cells and the related molecular mechanism

作　　者：李琳[1] 谢友红[2] Li Lin Xie Youhong(Department of Gastroenterology, The First Affiliated Hospital of Chongqing Medical University Center of Digestion,Affiliated University Town Hospital of Chongqing Medical University)

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016 [2]重庆医科大学附属大学城医院消化中心,重庆401331

出　　处：《重庆医科大学学报》2016年第10期1016-1021,共6页Journal of Chongqing Medical University

基　　金：重庆市渝中区科委资助项目(项目编号:X9237)

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Objective：To investigate the effect of transforming growth factor-α（TNF-α）on adipogenesis of SD rat bone marrow mesenchymal stem cells（BMSCs）and the related molecular mechanism. Methods：The well grown third-passage（P3）SD rat BMSCs were divided into three groups：control group,Ad group and Ad＋TNF-α group. RNA and proteins of the cells in three groups were collected at day 14. Cells were stained with oil red O on day 14 to examine the lipid accumulation in each group. Optical density for oil red O was determined by isopropanol extraction and was measured in a plate reader at 490 nm. The m RNA expressions of Wnt10 b,peroxisome proliferator-activated receptor-γ（PPAR-γ）,CCAAT/enhancer-binding protein-α（C/EBP-α）,fatty acid binding protein-4（FABP-4）and preadipocyte factor-1（Pref-1）were detected by q RT-PCR. The protein expression levels of P-β-catenin,PPAR-γ,C/EBP-α,FABP-4 were detected by Western blot. Results：The absorbance（A）value measured at 490 nm was significantly lower in Ad＋TNF-α group（0.360±0.035）than in Ad group（0.770±0.025）（P = 0.000）. q RT-PCR analysis showed that m RNA expressions of Wnt10 b and Pref-1 were significantly higher in Ad＋TNF-α group（17.050±2.706,4.135±0.280）than in Ad group（0. 575 ± 0. 065,0. 514 ± 0. 060）（P = 0. 02,P = 0. 006）.While the m RNA expressions of C/EBP-α,PPAR-γ and FABP-4 were significantly lower in Ad ＋TNF-α group（0.200 ±0.012,0.768 ±0.030,0.883 ±0.048） than in Ad group（2.965 ±0.455,2.330 ±0.211,3.847 ±0.171）（P =0.019,P =0.000,P =0.001）.Western blot showed the protein expressions of C/EBP-ɑ,PPAR-γ and FABP-4 were significantly lower in Ad＋TNF-α group（0.586±0.013,0.356±0.008,0.118±0.002）than in Ad group（1.082±0.018,0.840±0.112,1.094±0.038）（P=0.000）. The protein expression of P-β-catenin was significantly lower in Ad＋TNF-α group（0.648±0.005）than in Ad group（3.376±0.211）（P=0.000）. Conclusion ：The fact that TNF- α inhibits the termina

关 键 词：骨髓间充质干细胞 成脂分化 WNT/Β-CATENIN信号通路 前体脂肪细胞因子-1 

分 类 号：R364.5[医药卫生—病理学]