HIF-1α对DDAH1的调节及机制研究

发布时间：2018-06-10 11:13

本文选题：DDAH1 + HIF-1α　；参考：《华中科技大学》2011年硕士论文

【摘要】：研究目的:观察DDAH1在CoCl2干预下的表达变化,以及HIF-1α在DDAH1基因调控的作用机制。方法与结果:选取200g左右雄性wistar大鼠若干,随机分为2组分别为:CoCl2组大鼠腹腔注射CoCl2(30mg/kg)(n=8),NaCl组大鼠腹腔注射生理盐水(n=8)。我们发现CoCl2模拟缺氧刺激可以增加DDAH1蛋白的表达。细胞实验中我们发现用CoCl2干预的HUVEC、293T和HepG2中DDAH1的表达与HIF-1α有伴随效应。接下来的实验中我们利用HIF-1αSiRNA沉默HIF-1α蛋白的表达,实验的结果表明HIF-1α能够调节DDAH1的表达。对DDAH1启动子区的基因序列进行分析,我们发现有两个HRE元件,因此我们构建了DDAH1的启动子区不同片段长度的荧光报告基因的质粒,然后瞬时转染到HepG2和HEK293T中,CoCl2(200μM)干预8小时后我们检测荧光素酶活性,结果表明在DDAH1启动子-370?-387有可能是HIF-1α调控DDAH1启动子的活性区域。接着将上述质粒的HRE元件突变,即DDAH1启动子片段特定位点突变,转染到细胞,我们发现在诱导HIF-1α高表达的情况下荧光素酶活性不增加。同时,ChIP实验结果显示,HIF-1α能够特异性的结合到DDAH1的启动子区。结论:我们证明DDAH1是缺氧诱导基因,其转录是通过HIF-1α结合到大鼠DDAH1启动子的-370?-387的HRE元件来调控DDAH1的表达。
[Abstract]:Objective: to observe the changes of DDAH1 expression in CoCl2 and the mechanism of HIF-1 伪 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 伪. In the following experiment, we used HIF-1 伪 SiRNA to silence the expression of HIF-1 伪 protein. The results showed that HIF-1 伪 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 伪. 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 伪 expression was induced. At the same time, the results of ChIP showed that HIF-1 伪 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 伪 binding to rat DDAH1 promoter -370-387.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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