大鼠蛋白精氨酸甲基转移酶1短发夹RNA质粒的构建、鉴定及体外RNA干扰

发布时间：2018-06-27 20:57

  本文选题：蛋白精氨酸甲基转移酶1 + 基因干扰　； 参考：《山西医科大学》2008年硕士论文

【摘要】： 一、编码大鼠PRMT1基因shRNA质粒的构建和鉴定 目的:为研究蛋白精氨酸甲基转移酶1(PRMT1)基因表达的改变与血管内皮功能及动脉粥样硬化的关系,我们构建了PRMT1短发夹RNA质粒(pPRMT1-shRNA)。 方法:在GenBank中查到大鼠PRMT1基因mRNA序列并输入siRNA网上设计软件OptiRNA中,选取两条靶序列,设计并合成编码shRNA序列的DNA单链,经退火连接后,与双酶切线性化处理回收的pGenesil-1质粒载体连接,用含卡那霉素抗性的LB平板筛选阳性克隆,小提质粒后行酶切鉴定并测序。 结果:构建的大鼠pPRMT1-shRNA经酶切鉴定及测序与预期相符。 结论:本研究结果为进一步进行体外基因干扰PRMT1研究奠定了基础。 二、PRMT1-shRNA质粒转染大鼠主动脉内皮细胞条件的优化 目的:优化pPRMT1-shRNA在聚乙烯亚胺转染剂jetPEITM-RGD介导下转染原代培养大鼠主动脉内皮细胞的转染条件。 方法:贴块法原代培养大鼠主动脉内皮细胞,经免疫细胞化学法(S-P法)鉴定,取3～4代细胞,按不同jetPEITM-RGD/pPRMT1- shRNA比例进行转染,24小时后测定各组转染效率及细胞存活率。 结果:6孔培养板每孔加入3.0μg的pPRMT1 shRNA并以N/P=5加入jetPEITM-RGD转染效率最高,为53.54%。 结论:本研究为高效地进行细胞转染及进一步研究基因干扰PRMT1基因对血浆同型半胱氨酸(Hcy)、非对称性二甲基精氨酸(ADMA)水平和血管内皮功能的影响奠定了基础。 三、PRMT1-shRNA质粒对大鼠主动脉内皮细胞PRMT1表达的影响 目的:探讨pPRMT1-shRNA在体外对大鼠主动脉内皮细胞PRMT1基因mRNA表达水平的影响。 方法:用pPRMT1-shRNA1、pPRMT1-shRNA2阳性质粒、阴性对照pHK质粒转染大鼠主动脉内皮细胞,同时设立空白对照组。转染12、24小时后分别收集各组细胞,提取细胞总RNA,通过逆转录聚合酶链反应(RT-PCR)、2%琼脂糖凝胶电泳,凝胶成像分析系统分析各组PRMT1基因的mRNA表达情况。 结果:pPRMT1-shRNA1及pPRMT1-shRNA2转染组12、24小时PRMT1基因mRNA表达较阴性对照pHK及空白对照组均明显降低(P0.01);pPRMT1-shRNA1及pPRMT1- shRNA2转染组24小时PRMT1基因mRNA表达受抑较12小时显著(P0.05),且pPRMT1- shRNA1组比pPRMT1-shRNA2转染组受抑更显著(P0.05);而阴性对照pHK及空白对照组12及24小时PRMT1基因mRNA表达无明显差异,同一时间阴性对照pHK与空白对照组之间无明显差异。 结论:本研究所构建的pPRMT1-shRNA1及pPRMT1-shRNA2可在体外高效特异地抑制PRMT1的表达,为进一步的体内实验奠定了基础。
[Abstract]:1. Construction and identification of shRNA plasmid encoding rat PRMT1 gene. Objective: to study the relationship between the expression of protein arginine methyltransferase 1 (PRMT1) gene and vascular endothelial function and atherosclerosis. We constructed a short hairpin RNA plasmid (pPRMT1-shRNA). Methods: the mRNA sequence of rat PRMT1 gene was found in GenBank and was input into the siRNA web design software OptiRNA. Two target sequences were selected to design and synthesize the single strand of DNA encoding shRNA sequence. The plasmid vector pGenesil-1 was ligated with double digestion linearization. Positive clones were screened with kanamycin resistant LB plate. The plasmid was digested and sequenced. Results: the constructed rat pPRMT1-shRNA was confirmed by restriction endonuclease digestion and sequenced. Conclusion: this study lays a foundation for further study of gene interference PRMT1 in vitro. Objective: to optimize the transfection conditions of pPRMT1-shRNA into rat aortic endothelial cells mediated by jetPEITM-RGD. Objective: to optimize the transfection conditions of pPRMT1-shRNA in primary cultured rat aortic endothelial cells mediated by jetPEITM-RGD. Methods: the primary cultured rat aortic endothelial cells were identified by immunocytochemistry (S-P method). The 3G 4 passage cells were transfected with different jetPEITM-RGD / pPRMT1-shRNA ratio for 24 hours. The transfection efficiency and cell survival rate of each group were measured. Results addition of 3.0 渭 g pPRMT1 shRNA in each hole and addition of jetPEITM-RGD with N / Pn5 were the highest transfection efficiency (53.54). Conclusion: this study provides a basis for efficient cell transfection and further study on the effects of gene interference PRMT1 gene on plasma homocysteine (Hcy), asymmetric dimethyl arginine (ADMA) level and vascular endothelial function. Effect of PRMT1-shRNA plasmid on the expression of PRMT1 in rat aortic endothelial cells objective: to investigate the effect of pPRMT1-shRNA on the expression of PRMT1 mRNA in rat aortic endothelial cells in vitro. Methods: rat aortic endothelial cells were transfected with pPRMT1-shRNA1pPRMT1-shRNA2 positive plasmid, negative control pHK plasmid and blank control group. 24 hours after transfection, the cells were collected and the total RNAs were extracted. The mRNA expression of PRMT1 gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and 2% agarose gel electrophoresis. Results the expression of PRMT1 mRNA was significantly decreased in 1: pPRMT1-shRNA1 and pPRMT1-shRNA2 transfection groups compared with the negative control group (PHK) and the blank control group (P0.01). The expression of PRMT1 mRNA in the pPRMT1-shRNA1 and pPRMT1-shRNA2 transfection group was significantly inhibited than that in the pPRMT1-shRNA2 transfection group (P0.05), and the expression of PRMT1 mRNA in the pPRMT1-shRNA1 group was significantly lower than that in the pPRMT1-shRNA2 transfection group (P0.05). There was no significant difference in PRMT1 mRNA expression between the negative control group and the blank control group at 12 and 24 hours (P0.05). There was no significant difference between the negative control group and the blank control group at the same time. Conclusion: the pPRMT1-shRNA1 and pPRMT1-shRNA2 constructed in this study can inhibit the expression of PRMT1 in vitro and can lay a foundation for further in vivo experiments.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346;R541.4

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