p38α對人血管內(nèi)皮細胞一氧化氮合酶基因啟動子轉(zhuǎn)錄活性的調(diào)節(jié)
發(fā)布時間：2018-09-06 14:03
【摘要】： 目的：探討p38a對人血管內(nèi)皮細胞一氧化氮合酶(eNOS)基因啟動子轉(zhuǎn)錄活性的調(diào)節(jié)。方法：質(zhì)粒抽提試劑盒提取各種質(zhì)粒,包括pDsRedl-N1、pGL3-BASIC、pGL2-eNOS、pRL-TK、pcDNA3. p38α、及p38a (AF),并進行相應(yīng)的電泳鑒定和定量；用不同劑量的轉(zhuǎn)染試劑將紅色熒光蛋白載體pDsRedl-N1轉(zhuǎn)染HUVEC-12細胞,在熒光倒置DIC相差顯微鏡下通過冷CCD成像記錄轉(zhuǎn)染組紅色熒光蛋白載體pDsRedl-N1的發(fā)光以優(yōu)化轉(zhuǎn)染效率；建立雙熒光報告系統(tǒng),以pRL-TK為內(nèi)參照,將已經(jīng)構(gòu)建好的pGL2-eNOS質(zhì)粒分別與pGL3-B ASIC、pcDN A3、p38a及p38a(AF)載體共轉(zhuǎn)染HUVEC-12細胞,檢測各組細胞的eNOS基因啟動子轉(zhuǎn)錄活性；觀察p38信號通路特異性抑制劑SB203580對eNOS基因啟動子活性的影響,Western Blot分析磷酸化p38蛋白和非磷酸化p38蛋白的表達；利用siRNA干擾技術(shù)沉默p38a基因,進一步觀察eNOS基因啟動子活性的變化與p38蛋白表達的關(guān)系。結(jié)果：轉(zhuǎn)染p38a載體的細胞組其eNOS基因啟動子的活性降低,這種作用可以被其無活性誘變體p38a (AF)逆轉(zhuǎn)；抑制劑SB203580可導(dǎo)致磷酸化p38蛋白表達水平較對照組明顯降低,但非磷酸化p38蛋白表達水平基本保持不變,并且抑制劑SB203580阻止了eNOS基因啟動子活性的降低,其活性與轉(zhuǎn)染p38a載體的組相比具有明顯差異；不同濃度的siRNA對p38a的干擾效果不同,當(dāng)siRNA終濃度為100 nM時干擾效果最明顯,Western Blot顯示的條帶最弱；siRNA作用的時間不同,干擾效果也不同,當(dāng)濃度選定為100 nM時,作用48 h干擾效果最明顯；當(dāng)siRNA濃度為100nM作用48 h后,Western Blot顯示p38條帶明顯比其他組弱,而eNOS基因啟動子活性卻反而升高�？傊�,結(jié)果表明p38a的磷酸化可導(dǎo)致eNOS基因啟動子活性降低,該作用可被其無活性誘變體逆轉(zhuǎn),抑制劑SB203580和siRNA使p38a磷酸化水平降低,而eNOS基因啟動子活性反而升高。結(jié)論：以上結(jié)果提示p38α的活化可以下調(diào)eNOS基因啟動子活性。
[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 偽 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 偽 can down-regulate the promoter activity of eNOS gene.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341
本文編號：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 偽 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 偽 can down-regulate the promoter activity of eNOS gene.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341
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