激动TGR5对乳小鼠心肌成纤维细胞分泌胶原蛋白的影响及其机制-内科学专业论文.docxVIP

90％。4．2 90％。4．2 RT-PCR检测结果显示：cfs．v上TGR5 mRNA表达量较cfs明显 增多，P0．05。4．3 Western Blot结果显示：cfs．v内TGR5的蛋白含量较 cfs明显增多，PO．05。5、cAMP试剂盒检测胞内cAMP含量的结果显示： cfs—v+nomilin组细胞内cAMP含量较cfs组及cfs．v组均明显升高，P值均 0．05。6、ELISA试剂盒检测胶原蛋白I型和III型的结果显示： cfs．v+nomilin组细胞分泌的I型和III型胶原蛋白较cfs组及cfs—v组均明显 降低，P值均o．05。cfs．v+nomilin+BIRB．796组与cfs—v+nomilin组相比， 胶原蛋白I型和III的含量增多，P0．05。7、Western Blot法检测各组内 p-P38、Nrf2及HO．1的蛋白水平，结果显示：cfs—v+nomilin组细胞内p-P38、 Nrf2及HO一1的蛋白水平较cfs—v组均明显升高，P值均O．05。 cfs．v+nomilin+BIRB．796组的上述三种蛋白含量较cfs．v+nomilin组明显减 少，P均0．05。 结论：1、激动TGR5能减少乳小鼠心肌成纤维细胞分泌胶原蛋白I型 和III型。2、TGR5激活后可能通过P38途径引起Nrf2核转位启动HO．1 表达，抑制心肌成纤维细胞分泌胶原蛋白。3、P38．Nrf2信号通路可能是 TGR5参与抑制心肌纤维化的众多信号途径之一。 关键词：TGR5，胶原蛋白I型，胶原蛋白III型，心肌成纤维细胞， P38．MAPK，Nm．HO．1 万方数据 Effect Effect and mechanism of TGR5 activation on collagen secretion of cardiac fibroblasts in neonatal mice Abstract Objective：To study the effect of TGR5 on collagen secretion of cardiac fibroblasts in neonatal mice and explore the possible mechanism． Methods：1．The cultivation and identification of cardiac fibroblasts(cfs) of neonatal mice：some neonatal mices of 3 to 5 days were killed，the apex of the heats were taken out，digested by pancreatic enzyme and then cultured．Cfs were obtained through differential adhesion method and cultured for 7 to 1 0 days，then cells were subcultured and the third generation cells were identified by specific antibody fluorescent immuno assay．2．Detetcted the expression of TGR5 mRNA in cardiac fibroblasts of neonatal mice by Q—PCR：extract RNA from the third generation cardiac fibroblasts，at the same time RNA from heart， live and spleen were extracted as comparison．Then detect the concentration and purity of RNA．cDNA was synthesized by reverse transcription．the TGR5 primer was come frome Shanghai Biological Industry and Trade Co．，Ltd．And the target gene was detected by fluorescence ration PCR instrument．3．Design and pack TGR5 over expression lentivirus：the linearized vector was obtained by restriction enzyme and PCR amplified preparation