【摘要】 目的:研究转录因子Mip1对大鼠心肌细胞H9C2凋亡相关基因Bid的转录调控作用。方法:以H9C2细胞基因组DNA为模板,扩增Bid核心启动子片段,将其克隆入荧光素酶报告基因质粒PGL3-Basic中,构建重组载体,并采用双酶切法、PCR法及基因测序对其进行鉴定。用脂质体转染法将该载体转入Mip1不同程度过表达的H9C2细胞,检测该细胞Bid基因启动子区的转录活性。结果:成功克隆Bid基因启动子区,双酶切、PCR和基因测序均显示PGL3-Basic-Bidpromoter重组载体构建成功。荧光素酶相对活性检测显示在H9C2细胞中,随着Mip1转入的增多,Bid启动子区转录活性逐渐下降。结论:Mip1作为一个新的转录抑制因子,可以下调凋亡基因Bid的转录。更多还原

【Abstract】 Objective:To investigate the transcriptional regulation effects of Mip1 on the apoptosis-related gene Bid in H9C2 cells.Methods:The core promoter regions of Bid gene were amplified by nest PCR using rat genomic DNA as its template, cloned into luciferase reporter gene plasmid PGL3-Basic to construct restructured vector, and then identified by restriction digestion, PCR and gene sequencing.This Restructured vector was transfected into H9C2 cells with over-expressed Mip1 gene with Lipofectamine, and then the effects of Mip1 on the transcriptional activity of Bid gene promoter regions were detected in H9C2 cells.Results:The Bid gene promoter region was cloned and identified by enzyme digestion.Both PCR and gene sequencing showed that the recombinant vector PGL3-Basic-Bid was successfully constructed.The luciferase activity detection results indicated that the transcription activity of Bid promoter region gradually decreased in H9C2 cells with the transfered Mip1 increased.Conclusions:As a new transcriptional inhibited factor, Mip1 can down-regulate the transcription of apoptosis-related gene Bid.更多还原