本文選題：DDAH1 + HIF-1α��； 參考：《華中科技大學(xué)》2011年碩士論文

【摘要】：研究目的:觀察DDAH1在CoCl2干預(yù)下的表達變化,以及HIF-1α在DDAH1基因調(diào)控的作用機制。 方法與結(jié)果:選取200g左右雄性wistar大鼠若干,隨機分為2組分別為:CoCl2組大鼠腹腔注射CoCl2(30mg/kg)(n=8),NaCl組大鼠腹腔注射生理鹽水(n=8)。我們發(fā)現(xiàn)CoCl2模擬缺氧刺激可以增加DDAH1蛋白的表達。細胞實驗中我們發(fā)現(xiàn)用CoCl2干預(yù)的HUVEC、293T和HepG2中DDAH1的表達與HIF-1α有伴隨效應(yīng)。接下來的實驗中我們利用HIF-1αSiRNA沉默HIF-1α蛋白的表達,實驗的結(jié)果表明HIF-1α能夠調(diào)節(jié)DDAH1的表達。對DDAH1啟動子區(qū)的基因序列進行分析,我們發(fā)現(xiàn)有兩個HRE元件,因此我們構(gòu)建了DDAH1的啟動子區(qū)不同片段長度的熒光報告基因的質(zhì)粒,然后瞬時轉(zhuǎn)染到HepG2和HEK293T中,CoCl2(200μM)干預(yù)8小時后我們檢測熒光素酶活性,結(jié)果表明在DDAH1啟動子-370?-387有可能是HIF-1α調(diào)控DDAH1啟動子的活性區(qū)域。接著將上述質(zhì)粒的HRE元件突變,即DDAH1啟動子片段特定位點突變,轉(zhuǎn)染到細胞,我們發(fā)現(xiàn)在誘導(dǎo)HIF-1α高表達的情況下熒光素酶活性不增加。同時,ChIP實驗結(jié)果顯示,HIF-1α能夠特異性的結(jié)合到DDAH1的啟動子區(qū)。 結(jié)論:我們證明DDAH1是缺氧誘導(dǎo)基因,其轉(zhuǎn)錄是通過HIF-1α結(jié)合到大鼠DDAH1啟動子的-370?-387的HRE元件來調(diào)控DDAH1的表達。
[Abstract]:Objective: to observe the changes of DDAH1 expression in CoCl2 and the mechanism of HIF-1 偽 in DDAH1 gene regulation. Methods and results: several male wistar rats were selected. Two groups were randomly divided into two groups: the rats in the control group were injected with 30 mg / kg of CoCl2 and 30 mg / kg, respectively, and the rats in the NaCl group were injected intraperitoneally with normal saline. We found that the expression of DDAH1 protein was increased by simulated hypoxia stimulation by CoCl2. We found that the expression of DDAH1 in HUVEC-293T and HepG2 induced by CoCl2 was associated with HIF-1 偽. In the following experiment, we used HIF-1 偽 SiRNA to silence the expression of HIF-1 偽 protein. The results showed that HIF-1 偽 could regulate the expression of DDAH1. By analyzing the gene sequence of DDAH1 promoter region, we found that there are two HRE elements, so we constructed the plasmid of fluorescent reporter gene with different fragment length in the promoter region of DDAH1. The luciferase activity was detected after 8 hours of transient transfection into HepG2 and HEK293T. The results showed that the DDAH1 promoter -370-387 might be the active region of DDAH1 promoter regulated by HIF-1 偽. Then the HRE element mutation of the above plasmid, that is, the specific site mutation of DDAH1 promoter fragment, was transfected into the cells. We found that luciferase activity did not increase when HIF-1 偽 expression was induced. At the same time, the results of ChIP showed that HIF-1 偽 could specifically bind to the promoter region of DDAH1.Conclusion: we suggest that DDAH1 is anoxia-induced gene, and its transcription is regulated by HRE element of HIF-1 偽 binding to rat DDAH1 promoter -370-387.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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