人CD2AP基因启动子的鉴定及其功能研究

发布时间：2018-06-20 19:37

  本文选题：CD2相关蛋白 + 启动子　； 参考：《南京医科大学》2010年博士论文

【摘要】： 目的: 鉴定与分析人CD2AP(CD2-associated protein,CD2AP)基因启动子的结构与功能,研究其调控的分子机制,为探讨CD2AP在相关肾脏病发病中的作用积累基础研究。 方法: 采用5'-RACE法确定人CD2AP基因的转录起始位点,利用PCR进行逐段缺失突变,克隆其5'侧翼DNA序列,构建一系列荧光素酶报告载体,转染人肾小管上皮细胞等细胞系,确定CD2AP启动子的核苷酸范围。利用点突变技术、凝胶迁移率改变试验、染色质免疫沉淀、小RNA干扰和基因过表达等实验,分析CD2AP启动子区的转录因子结合位点。观察过氧化氢(H2O2)模拟氧化应激状态下CD2AP启动子的活性变化,以及血管内皮生长因子对CD2AP启动子活性的影响。转染CD2AP启动子荧光素酶报告质粒至人胚肾293细胞,研究呼吸道合胞病毒感染对CD2AP启动子活性的影响与机制。 结果: 第一部分:确定人CD2AP基因启动子位于转录起始位点上游-558bp至-1bp之间,一个CREB和两个SP1转录因子结合位点维持其基本转录活性。 第二部分:证实在人肾小管上皮细胞中,表皮生长因子(epidermal growth factor,EGF)可以募集AP-1家族的JunD和c-fos成员形成复合体与CD2AP启动子区的一个AP-1样位点(5-TGAGCTCA-3)相结合,激活CD2AP启动子的转录活性,并对血管紧张素Ⅱ诱导的人肾小管上皮细胞凋亡产生拮抗作用。 第三部分:证实在氧化应激状态下,CD2AP启动子活性下调,CD2AP与F-actin在细胞内的共定位减少。而低剂量的血管内皮生长因子可以拮抗H2O2诱导的5CD2AP启动子活性下调。 第四部分:证实呼吸道合胞病毒感染早期激活CD2AP启动子活性,晚期则抑制其活化。AP-1家族的JunD参与呼吸道合胞病毒感染早期CD2AP启动子的活性调节。而呼吸道合胞病毒感染晚期CD2AP启动子活性的抑制至少部分依赖于RIG-1/MAVS信号的传导。 结论: 人CD2AP基因启动子位于转录起始位点上游-558至-1bp处,转录因子CREB和SP1维持其基本转录活性。EGF/AP-1/CD2AP启动子是一个促人肾小管上皮细胞生存的信号途径。低剂量的血管内皮生长因子拮抗氧化应激诱导的CD2AP启动子活性下调,可能是其肾脏保护作用的原因之一。呼吸道合胞病毒感染对CD2AP启动子活性的调节有双向作用。呼吸道合胞病毒感染后期造成的CD2AP启动子转录活性显著下调,可能是其产生肾脏损害的机制之一。
[Abstract]:Aim: to identify and analyze the structure and function of the promoter of human CD2APP CD2-associated protein (CD2AP) gene, and to study the molecular mechanism of CD2AP in order to investigate the role of CD2AP in the pathogenesis of renal diseases. Methods: the transcriptional initiation site of human CD2AP gene was determined by 5 '-RACE method. A series of luciferase report vectors were constructed by cloning its 5'flanking DNA sequence by PCR. The nucleotide range of CD2 AP promoter was determined by transfection of human renal tubular epithelial cells and other cell lines. The transcription factor binding sites of CD2AP promoter were analyzed by point mutation, gel mobility change test, chromatin immunoprecipitation, small RNA interference and gene overexpression. The activity of CD2AP promoter and the effect of vascular endothelial growth factor (VEGF) on the activity of CD2AP promoter were observed. CD2AP promoter luciferase reporter plasmid was transfected into human embryonic kidney 293 cells to study the effect and mechanism of respiratory syncytial virus infection on CD2AP promoter activity. Results: in the first part, the promoter of human CD2AP gene was located between -558bp and -1bp upstream of the transcription initiation site. One CREB and two SP1 transcription factor binding sites maintained their basic transcriptional activity. Part two: it is proved that epidermal growth factor (EGF) can activate the transcriptional activity of CD2AP promoter by recruiting JunD and c-fos member forming complex of AP-1 family and binding to an AP-1 like site of CD2AP promoter, 5-TGAGCTCA-3, in human renal tubular epithelial cells. It also antagonized the apoptosis of human renal tubular epithelial cells induced by angiotensin 鈪，

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