p38α对人血管内皮细胞一氧化氮合酶基因启动子转录活性的调节
发布时间：2018-09-06 14:03
【摘要】： 目的：探讨p38a对人血管内皮细胞一氧化氮合酶(eNOS)基因启动子转录活性的调节。方法：质粒抽提试剂盒提取各种质粒,包括pDsRedl-N1、pGL3-BASIC、pGL2-eNOS、pRL-TK、pcDNA3. p38α、及p38a (AF),并进行相应的电泳鉴定和定量；用不同剂量的转染试剂将红色荧光蛋白载体pDsRedl-N1转染HUVEC-12细胞,在荧光倒置DIC相差显微镜下通过冷CCD成像记录转染组红色荧光蛋白载体pDsRedl-N1的发光以优化转染效率；建立双荧光报告系统,以pRL-TK为内参照,将已经构建好的pGL2-eNOS质粒分别与pGL3-B ASIC、pcDN A3、p38a及p38a(AF)载体共转染HUVEC-12细胞,检测各组细胞的eNOS基因启动子转录活性；观察p38信号通路特异性抑制剂SB203580对eNOS基因启动子活性的影响,Western Blot分析磷酸化p38蛋白和非磷酸化p38蛋白的表达；利用siRNA干扰技术沉默p38a基因,进一步观察eNOS基因启动子活性的变化与p38蛋白表达的关系。结果：转染p38a载体的细胞组其eNOS基因启动子的活性降低,这种作用可以被其无活性诱变体p38a (AF)逆转；抑制剂SB203580可导致磷酸化p38蛋白表达水平较对照组明显降低,但非磷酸化p38蛋白表达水平基本保持不变,并且抑制剂SB203580阻止了eNOS基因启动子活性的降低,其活性与转染p38a载体的组相比具有明显差异；不同浓度的siRNA对p38a的干扰效果不同,当siRNA终浓度为100 nM时干扰效果最明显,Western Blot显示的条带最弱；siRNA作用的时间不同,干扰效果也不同,当浓度选定为100 nM时,作用48 h干扰效果最明显；当siRNA浓度为100nM作用48 h后,Western Blot显示p38条带明显比其他组弱,而eNOS基因启动子活性却反而升高。总之,结果表明p38a的磷酸化可导致eNOS基因启动子活性降低,该作用可被其无活性诱变体逆转,抑制剂SB203580和siRNA使p38a磷酸化水平降低,而eNOS基因启动子活性反而升高。结论：以上结果提示p38α的活化可以下调eNOS基因启动子活性。
[Abstract]:Aim: to investigate the effect of p38a on the transcriptional activity of nitric oxide synthase (eNOS) gene promoter in human vascular endothelial cells (VEC). Methods: plasmid extraction kit was used to extract various plasmids, including pDsRedl-N1,pGL3-BASIC,pGL2-eNOS,pRL-TK,pcDNA3. p38 伪 and p38a (AF), and to identify and quantify them by electrophoresis. The red fluorescent protein vector pDsRedl-N1 was transfected into HUVEC-12 cells with different doses of transfection reagent. The luminescence of red fluorescent protein vector pDsRedl-N1 in transfection group was recorded by cold CCD imaging under fluorescence inverted DIC phase contrast microscope to optimize the transfection efficiency. A double fluorescence report system was established with pRL-TK as internal reference. The constructed pGL2-eNOS plasmids were cotransfected with pGL3-B ASIC,pcDN A3G p38a and p38a (AF) vectors into HUVEC-12 cells to detect the transcriptional activity of eNOS gene promoter. Effects of p38 signal Pathway specific inhibitor SB203580 on the Promoter activity of eNOS Gene the expression of phosphorylated p38 protein and non-phosphorylated p38 protein was analyzed by Western Blot, and the p38a gene was silenced by siRNA interference technique. To investigate the relationship between the activity of eNOS promoter and the expression of p38 protein. Results: the activity of eNOS gene promoter was decreased in p38a vector transfected cell group, which could be reversed by p38a (AF), and the expression level of phosphorylated p38 protein was significantly decreased by inhibitor SB203580. However, the expression level of non-phosphorylated p38 protein remained basically unchanged, and the inhibitor SB203580 prevented the activity of eNOS gene promoter from decreasing, and its activity was significantly different from that of p38a vector transfection group, and the interference effect of different concentrations of siRNA on p38a was different. When the final concentration of siRNA was 100 nM, the interference effect was the most obvious. When the final concentration of siRNA was 100 nM, the interference time was different and the interference effect was different. When the concentration was 100 nM, the interference effect was the most obvious at 48 h. When siRNA was treated with 100nM for 48 h, the p38 band was weaker than that in other groups, but the activity of eNOS gene promoter was increased. In conclusion, the phosphorylation of p38a resulted in a decrease in the activity of eNOS gene promoter, which could be reversed by its inactive mutagens. The phosphorylation level of p38a was decreased by inhibitors SB203580 and siRNA, while the activity of eNOS gene promoter increased. Conclusion: these results suggest that the activation of p38 伪 can down-regulate the promoter activity of eNOS gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
本文编号：2226576
[Abstract]:Aim: to investigate the effect of p38a on the transcriptional activity of nitric oxide synthase (eNOS) gene promoter in human vascular endothelial cells (VEC). Methods: plasmid extraction kit was used to extract various plasmids, including pDsRedl-N1,pGL3-BASIC,pGL2-eNOS,pRL-TK,pcDNA3. p38 伪 and p38a (AF), and to identify and quantify them by electrophoresis. The red fluorescent protein vector pDsRedl-N1 was transfected into HUVEC-12 cells with different doses of transfection reagent. The luminescence of red fluorescent protein vector pDsRedl-N1 in transfection group was recorded by cold CCD imaging under fluorescence inverted DIC phase contrast microscope to optimize the transfection efficiency. A double fluorescence report system was established with pRL-TK as internal reference. The constructed pGL2-eNOS plasmids were cotransfected with pGL3-B ASIC,pcDN A3G p38a and p38a (AF) vectors into HUVEC-12 cells to detect the transcriptional activity of eNOS gene promoter. Effects of p38 signal Pathway specific inhibitor SB203580 on the Promoter activity of eNOS Gene the expression of phosphorylated p38 protein and non-phosphorylated p38 protein was analyzed by Western Blot, and the p38a gene was silenced by siRNA interference technique. To investigate the relationship between the activity of eNOS promoter and the expression of p38 protein. Results: the activity of eNOS gene promoter was decreased in p38a vector transfected cell group, which could be reversed by p38a (AF), and the expression level of phosphorylated p38 protein was significantly decreased by inhibitor SB203580. However, the expression level of non-phosphorylated p38 protein remained basically unchanged, and the inhibitor SB203580 prevented the activity of eNOS gene promoter from decreasing, and its activity was significantly different from that of p38a vector transfection group, and the interference effect of different concentrations of siRNA on p38a was different. When the final concentration of siRNA was 100 nM, the interference effect was the most obvious. When the final concentration of siRNA was 100 nM, the interference time was different and the interference effect was different. When the concentration was 100 nM, the interference effect was the most obvious at 48 h. When siRNA was treated with 100nM for 48 h, the p38 band was weaker than that in other groups, but the activity of eNOS gene promoter was increased. In conclusion, the phosphorylation of p38a resulted in a decrease in the activity of eNOS gene promoter, which could be reversed by its inactive mutagens. The phosphorylation level of p38a was decreased by inhibitors SB203580 and siRNA, while the activity of eNOS gene promoter increased. Conclusion: these results suggest that the activation of p38 伪 can down-regulate the promoter activity of eNOS gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
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