myocardin调控心肌l型ca2通道转录机制研究生物学专业论文.docxVIP

细胞免疫荧光技术检测LTCC基因蛋白表达量增加且与对照组相比更多的分布在 细胞免疫荧光技术检测LTCC基因蛋白表达量增加且与对照组相比更多的分布在 细胞核和胞浆中；干扰内源性Myocardin之后结果与之相反。 3．Myocardin激活LTCC Cavl．2基因的转录依赖其启动子上的CarGbox： LTCC基因启动子区CarGbox基因突变后，报告基因活性显著下降；Chip检 测结果显示：Myocardin与LTCC Cavl．2启动子区CarGbox特异性结合。 结论：Myocardin通过与LTCC CavI．2基因启动子区CarGbox结合进而激活 其转录和表达，影响钙离子通道蛋白的分布，促进Ca2+内流，增加膜电流。 关键词：Myocardin；钙离子通道；转录因子；转录调控；启动子 II 万方数据 Abstractobjective：The Abstract objective：The transcriptional regulation of L—Ca什channel mediated by Myocardin． Methods： 1．Demonstrating whether the activation of Cavl．2 LTCC gene mediated by Myocardin would affect the Cardiomyocyte membrane Ca+current Primary cardiocytes were infected with LV-Myocardin lentiviral plasmid to over -express Myocardin or LV-sh-Myocardin lentiviral plasmid to interfer Myocardin or blank lentivirus plasmid PTK642 as contr01．48 h lateL Cardiomyocyte membrane Ca+ current was detected by electrophysiological patch clamp technique． 2．Proving that Myocardin can activate expression of the LTCC gene： Cardiomyocyte H9C2 was infected with LV-Myocardin lentiviral plasmid to over —express Myocardin．48 h later,we used RT-PCR and real—time PCR to detect the level of the LTCC mRNA；The protein expression level of LTCC WaS detected by Western blotting；The distribution of LTCC protein was detected by immune fluorescence． 3．Proving that the interference of Myocardin Can inhibit the expression of LTCC Cav 1．2 gene： Cardiomyocyte H9C2 was infected with LV-sh—Myocardin lentiviral plasmid to interfer Myocardin．48 h later,we used RT-PCR and real—time PCR to detect the level of LTCC mRNA；The protein expression level of LTCC Was detected by Western blotting； The distribution of LTCC。protein was detected by immune fluorescence． 4．Proving that Myocardin Can activate the transcription ofthe LTCC gene： Co—transfect Myocardin and Cav 1．2 luciferase reporter plasmid into cultured 293-T cells，using luciferase aSsay to detect the promoter activity of Cavl．2． 5．Demonstrating whether the activation of LTCC Cav 1．2 g