低氧诱导因子-1对肝细胞生长因子基因表达调控机制的研究

发布时间：2018-06-08 05:50

本文选题：低氧 + 低氧诱导因子-1　；参考：《中国协和医科大学》2006年博士论文

【摘要】：目的：第一部分：(1) 观察氯化钴(CoCl_2)及HIF-1α过表达对乳鼠心肌细胞及人宫颈癌Hela细胞存活及凋亡状况的影响。(2) 观察CoCl_2模拟的低氧以及由pcDNA3-HIF-1α真核表达质粒转染介导的HIF-1α过表达对乳鼠心肌细胞和Hela细胞中HGF／c-Met系统mRNA和蛋白表达的调节。(3)对人HGF基因启动子区进行生物信息学分析，寻找可能介导HGF低氧表达调控的转录因子作用位点。第二部分：根据生物信息学分析结果，在Hela和转染效率较高HEK293细胞中，研究HIF-1α对TGF-β2基因表达调控的作用机制，并进一步观察重组转化生长因子-β2(transforming growth factor-β2，，TGF-β2)对Hela细胞中HGF表达的影响，方法：第一部分：(1) 体外培养乳鼠原代心肌细胞和Hela细胞，应用MTT和流式细胞仪检测CoCl_2对乳鼠心肌细胞和Hela细胞存活、增殖及凋亡的影响。(2) 构建pcDNA3-HIF-1α真核表达质粒(3) 免疫印迹检测CoCl_2和pcDNA3-HIF-1α质粒转染诱导的HIF-1α过表达后，Hela细胞中凋亡相关蛋白Bc1-2和Bax的表达。(4)RT-PCR检测100μmol／L CoCl_2作用6～72h后，心肌细胞中HGF mRNA的表达。(5)Western Blot检测100gmol／L CoCl_2作用心肌细胞12h后HIF-1α蛋白的表达。(6) 相对荧光实时定量PCR检测CoCl_2和pcDNA3-HIF-1α质粒转染后，Hela细胞中HGF／c-Met mRNA的表达。(7) ELISA检测CoCl_2对Hela细胞中分泌性HGF蛋白表达的影响。(8) 利用生物信息学分析人HGF基因启动子区可能的转录因子结合位点。第二部分：(1) 构建pGL3-EPO-HRE荧光素酶报告质粒和pGL3-TGF-β2及其五个突变体的荧光素酶报告质粒。(2) Western Blot和双荧光素酶报告基因法检测CoCl_2和pcDNA3-HIF-1α质粒转染后，Hela和HEK293细胞中HIF-1α蛋白的表达量及其DNA结合活性。(3) 相对荧光实时定量PCR检测250μmol／L CoCl_2单独作用Hela和HEK293细胞6h后，TGF-β2mRNA的表达，以及CoCl_2处理和pcDNA3-HIF-1α质
[Abstract]:Objective: to observe the effects of CoCl2) and HIF-1 伪 overexpression on the survival and apoptosis of neonatal rat cardiomyocytes and human cervical cancer Hela cells. Part 1: 1) to observe the simulated hypoxia of CoCl2 and the overexpression of HIF-1 伪 mediated by pcDNA3-HIF-1 伪 eukaryotic expression plasmid. The expression of HGF / c-Met mRNA and protein in neonatal rat cardiomyocytes and Hela cells were analyzed by bioinformatics. To search for transcription factor action sites that may mediate the regulation of hypoxia expression of HGF. Part two: according to the results of bioinformatics analysis, we studied the mechanism of HIF-1 伪 regulating TGF- 尾 2 gene expression in Hela cells and HEK293 cells with high transfection efficiency. The effects of recombinant transforming growth factor- 尾 2(transforming growth factor- 尾 2 (TGF- 尾 2) on the expression of HGF- 尾 2 in Hela cells were further investigated. Methods: the first part: primary cultured neonatal rat cardiomyocytes and Hela cells were cultured in vitro. MTT and flow cytometry were used to detect the survival of CoCl2 in neonatal rat cardiomyocytes and Hela cells. The expression of apoptosis-related proteins Bc1-2 and Bax in Hela cells induced by transfection of CoCl2 and pcDNA3-HIF-1 伪 was detected by Western blot. The expression of apoptosis-related proteins Bc1-2 and Bax in Hela cells was detected by RT-PCR. After treatment with 100 渭 mol / L CoCl2, the expression of apoptosis-related proteins Bc1-2 and Bax in Hela cells was detected after treatment with 100 渭 mol / L CoCl2. Expression of HGF mRNA in cardiomyocytes. Western Blot to detect HIF-1 伪 protein expression in cardiomyocytes treated with 100 g mol / L CoCl2 for 12 h.) relative real-time quantitative PCR was used to detect the expression of HGF- 尾 -Met mRNA in Hela cells after transfection of Cocl2 and pcDNA3-HIF-1 伪 plasmids.) Elisa was used to detect the expression of HGF / c-Met mRNA in Hela cells by CoCl2 and pcDNA3-HIF-1 伪 plasmid transfection. The possible transcription factor binding sites in the promoter region of human HGF gene were analyzed by bioinformatics. Part 2: 1) Construction of pGL3-EPO-HRE luciferase reporter plasmid and pGL3-TGF- 尾 2 and its five mutants. The expression of HIF-1 伪 protein in Hela and HEK293 cells after transfection with CoCl2 and pcDNA3-HIF-1 伪 plasmids was detected by Western Blot and double luciferase reporter gene method. The expression of TGF- 尾 2 mRNA in Hela and HEK293 cells was detected by relative fluorescence real-time quantitative PCR after exposure to 250 渭 mol / L CoCl2 for 6 h. CoCl2 treatment and pcDNA3-HIF-1 伪
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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