本文選題：CD2相關(guān)蛋白 + 啟動(dòng)子　； 參考：《南京醫(yī)科大學(xué)》2010年博士論文

【摘要】： 目的: 鑒定與分析人CD2AP(CD2-associated protein,CD2AP)基因啟動(dòng)子的結(jié)構(gòu)與功能,研究其調(diào)控的分子機(jī)制,為探討CD2AP在相關(guān)腎臟病發(fā)病中的作用積累基礎(chǔ)研究。 方法: 采用5'-RACE法確定人CD2AP基因的轉(zhuǎn)錄起始位點(diǎn),利用PCR進(jìn)行逐段缺失突變,克隆其5'側(cè)翼DNA序列,構(gòu)建一系列熒光素酶報(bào)告載體,轉(zhuǎn)染人腎小管上皮細(xì)胞等細(xì)胞系,確定CD2AP啟動(dòng)子的核苷酸范圍。利用點(diǎn)突變技術(shù)、凝膠遷移率改變?cè)囼?yàn)、染色質(zhì)免疫沉淀、小RNA干擾和基因過表達(dá)等實(shí)驗(yàn),分析CD2AP啟動(dòng)子區(qū)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。觀察過氧化氫(H2O2)模擬氧化應(yīng)激狀態(tài)下CD2AP啟動(dòng)子的活性變化,以及血管內(nèi)皮生長因子對(duì)CD2AP啟動(dòng)子活性的影響。轉(zhuǎn)染CD2AP啟動(dòng)子熒光素酶報(bào)告質(zhì)粒至人胚腎293細(xì)胞,研究呼吸道合胞病毒感染對(duì)CD2AP啟動(dòng)子活性的影響與機(jī)制。 結(jié)果: 第一部分:確定人CD2AP基因啟動(dòng)子位于轉(zhuǎn)錄起始位點(diǎn)上游-558bp至-1bp之間,一個(gè)CREB和兩個(gè)SP1轉(zhuǎn)錄因子結(jié)合位點(diǎn)維持其基本轉(zhuǎn)錄活性。 第二部分:證實(shí)在人腎小管上皮細(xì)胞中,表皮生長因子(epidermal growth factor,EGF)可以募集AP-1家族的JunD和c-fos成員形成復(fù)合體與CD2AP啟動(dòng)子區(qū)的一個(gè)AP-1樣位點(diǎn)(5-TGAGCTCA-3)相結(jié)合,激活CD2AP啟動(dòng)子的轉(zhuǎn)錄活性,并對(duì)血管緊張素Ⅱ誘導(dǎo)的人腎小管上皮細(xì)胞凋亡產(chǎn)生拮抗作用。 第三部分:證實(shí)在氧化應(yīng)激狀態(tài)下,CD2AP啟動(dòng)子活性下調(diào),CD2AP與F-actin在細(xì)胞內(nèi)的共定位減少。而低劑量的血管內(nèi)皮生長因子可以拮抗H2O2誘導(dǎo)的5CD2AP啟動(dòng)子活性下調(diào)。 第四部分:證實(shí)呼吸道合胞病毒感染早期激活CD2AP啟動(dòng)子活性,晚期則抑制其活化。AP-1家族的JunD參與呼吸道合胞病毒感染早期CD2AP啟動(dòng)子的活性調(diào)節(jié)。而呼吸道合胞病毒感染晚期CD2AP啟動(dòng)子活性的抑制至少部分依賴于RIG-1/MAVS信號(hào)的傳導(dǎo)。 結(jié)論: 人CD2AP基因啟動(dòng)子位于轉(zhuǎn)錄起始位點(diǎn)上游-558至-1bp處,轉(zhuǎn)錄因子CREB和SP1維持其基本轉(zhuǎn)錄活性。EGF/AP-1/CD2AP啟動(dòng)子是一個(gè)促人腎小管上皮細(xì)胞生存的信號(hào)途徑。低劑量的血管內(nèi)皮生長因子拮抗氧化應(yīng)激誘導(dǎo)的CD2AP啟動(dòng)子活性下調(diào),可能是其腎臟保護(hù)作用的原因之一。呼吸道合胞病毒感染對(duì)CD2AP啟動(dòng)子活性的調(diào)節(jié)有雙向作用。呼吸道合胞病毒感染后期造成的CD2AP啟動(dòng)子轉(zhuǎn)錄活性顯著下調(diào),可能是其產(chǎn)生腎臟損害的機(jī)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，

本文編號(hào)：2045442