本文選題：低氧 + 低氧誘導(dǎo)因子-1　； 參考：《中國協(xié)和醫(yī)科大學(xué)》2006年博士論文

【摘要】：目的：第一部分：(1) 觀察氯化鈷(CoCl_2)及HIF-1α過表達對乳鼠心肌細胞及人宮頸癌Hela細胞存活及凋亡狀況的影響。(2) 觀察CoCl_2模擬的低氧以及由pcDNA3-HIF-1α真核表達質(zhì)粒轉(zhuǎn)染介導(dǎo)的HIF-1α過表達對乳鼠心肌細胞和Hela細胞中HGF／c-Met系統(tǒng)mRNA和蛋白表達的調(diào)節(jié)。(3)對人HGF基因啟動子區(qū)進行生物信息學(xué)分析，尋找可能介導(dǎo)HGF低氧表達調(diào)控的轉(zhuǎn)錄因子作用位點。第二部分：根據(jù)生物信息學(xué)分析結(jié)果，在Hela和轉(zhuǎn)染效率較高HEK293細胞中，研究HIF-1α對TGF-β2基因表達調(diào)控的作用機制，并進一步觀察重組轉(zhuǎn)化生長因子-β2(transforming growth factor-β2，，TGF-β2)對Hela細胞中HGF表達的影響， 方法：第一部分：(1) 體外培養(yǎng)乳鼠原代心肌細胞和Hela細胞，應(yīng)用MTT和流式細胞儀檢測CoCl_2對乳鼠心肌細胞和Hela細胞存活、增殖及凋亡的影響。(2) 構(gòu)建pcDNA3-HIF-1α真核表達質(zhì)粒(3) 免疫印跡檢測CoCl_2和pcDNA3-HIF-1α質(zhì)粒轉(zhuǎn)染誘導(dǎo)的HIF-1α過表達后，Hela細胞中凋亡相關(guān)蛋白Bc1-2和Bax的表達。(4)RT-PCR檢測100μmol／L CoCl_2作用6～72h后，心肌細胞中HGF mRNA的表達。(5)Western Blot檢測100gmol／L CoCl_2作用心肌細胞12h后HIF-1α蛋白的表達。(6) 相對熒光實時定量PCR檢測CoCl_2和pcDNA3-HIF-1α質(zhì)粒轉(zhuǎn)染后，Hela細胞中HGF／c-Met mRNA的表達。(7) ELISA檢測CoCl_2對Hela細胞中分泌性HGF蛋白表達的影響。(8) 利用生物信息學(xué)分析人HGF基因啟動子區(qū)可能的轉(zhuǎn)錄因子結(jié)合位點。第二部分：(1) 構(gòu)建pGL3-EPO-HRE熒光素酶報告質(zhì)粒和pGL3-TGF-β2及其五個突變體的熒光素酶報告質(zhì)粒。(2) Western Blot和雙熒光素酶報告基因法檢測CoCl_2和pcDNA3-HIF-1α質(zhì)粒轉(zhuǎn)染后，Hela和HEK293細胞中HIF-1α蛋白的表達量及其DNA結(jié)合活性。(3) 相對熒光實時定量PCR檢測250μmol／L CoCl_2單獨作用Hela和HEK293細胞6h后，TGF-β2mRNA的表達，以及CoCl_2處理和pcDNA3-HIF-1α質(zhì)
[Abstract]:Objective: to observe the effects of CoCl2) and HIF-1 偽 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 偽 mediated by pcDNA3-HIF-1 偽 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 偽 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 偽 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 偽 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 偽 plasmids.) Elisa was used to detect the expression of HGF / c-Met mRNA in Hela cells by CoCl2 and pcDNA3-HIF-1 偽 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 偽 protein in Hela and HEK293 cells after transfection with CoCl2 and pcDNA3-HIF-1 偽 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 偽
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R363

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