發(fā)布時間：2018-06-27 20:57

  本文選題：蛋白精氨酸甲基轉移酶1 + 基因干擾�。� 參考：《山西醫(yī)科大學》2008年碩士論文

【摘要】： 一、編碼大鼠PRMT1基因shRNA質粒的構建和鑒定 目的:為研究蛋白精氨酸甲基轉移酶1(PRMT1)基因表達的改變與血管內皮功能及動脈粥樣硬化的關系,我們構建了PRMT1短發(fā)夾RNA質粒(pPRMT1-shRNA)。 方法:在GenBank中查到大鼠PRMT1基因mRNA序列并輸入siRNA網上設計軟件OptiRNA中,選取兩條靶序列,設計并合成編碼shRNA序列的DNA單鏈,經退火連接后,與雙酶切線性化處理回收的pGenesil-1質粒載體連接,用含卡那霉素抗性的LB平板篩選陽性克隆,小提質粒后行酶切鑒定并測序。 結果:構建的大鼠pPRMT1-shRNA經酶切鑒定及測序與預期相符。 結論:本研究結果為進一步進行體外基因干擾PRMT1研究奠定了基礎。 二、PRMT1-shRNA質粒轉染大鼠主動脈內皮細胞條件的優(yōu)化 目的:優(yōu)化pPRMT1-shRNA在聚乙烯亞胺轉染劑jetPEITM-RGD介導下轉染原代培養(yǎng)大鼠主動脈內皮細胞的轉染條件。 方法:貼塊法原代培養(yǎng)大鼠主動脈內皮細胞,經免疫細胞化學法(S-P法)鑒定,取3～4代細胞,按不同jetPEITM-RGD/pPRMT1- shRNA比例進行轉染,24小時后測定各組轉染效率及細胞存活率。 結果:6孔培養(yǎng)板每孔加入3.0μg的pPRMT1 shRNA并以N/P=5加入jetPEITM-RGD轉染效率最高,為53.54%。 結論:本研究為高效地進行細胞轉染及進一步研究基因干擾PRMT1基因對血漿同型半胱氨酸(Hcy)、非對稱性二甲基精氨酸(ADMA)水平和血管內皮功能的影響奠定了基礎。 三、PRMT1-shRNA質粒對大鼠主動脈內皮細胞PRMT1表達的影響 目的:探討pPRMT1-shRNA在體外對大鼠主動脈內皮細胞PRMT1基因mRNA表達水平的影響。 方法:用pPRMT1-shRNA1、pPRMT1-shRNA2陽性質粒、陰性對照pHK質粒轉染大鼠主動脈內皮細胞,同時設立空白對照組。轉染12、24小時后分別收集各組細胞,提取細胞總RNA,通過逆轉錄聚合酶鏈反應(RT-PCR)、2%瓊脂糖凝膠電泳,凝膠成像分析系統(tǒng)分析各組PRMT1基因的mRNA表達情況。 結果:pPRMT1-shRNA1及pPRMT1-shRNA2轉染組12、24小時PRMT1基因mRNA表達較陰性對照pHK及空白對照組均明顯降低(P0.01);pPRMT1-shRNA1及pPRMT1- shRNA2轉染組24小時PRMT1基因mRNA表達受抑較12小時顯著(P0.05),且pPRMT1- shRNA1組比pPRMT1-shRNA2轉染組受抑更顯著(P0.05);而陰性對照pHK及空白對照組12及24小時PRMT1基因mRNA表達無明顯差異,同一時間陰性對照pHK與空白對照組之間無明顯差異。 結論:本研究所構建的pPRMT1-shRNA1及pPRMT1-shRNA2可在體外高效特異地抑制PRMT1的表達,為進一步的體內實驗奠定了基礎。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346;R541.4

【相似文獻】

相關博士學位論文 前1條

1 王猛;ANXA2在視網膜新生血管形成過程中的表達及作用機制[D];青島大學;2012年

相關碩士學位論文 前8條

1 方雅琴;大鼠蛋白精氨酸甲基轉移酶1短發(fā)夾RNA質粒的構建、鑒定及體外RNA干擾[D];山西醫(yī)科大學;2008年

2 徐廣芬;缺氧誘導因子-1α在皮膚鱗狀細胞癌中的表達及促進細胞增殖作用[D];華中科技大學;2011年

3 張福軍;Bcl-2沉默促進多藥抗性口腔腺樣囊性癌細胞株細胞凋亡作用的實驗研究[D];重慶醫(yī)科大學;2006年

4 豐菁;RNAi沉默ADAMs家族基因對腫瘤細胞生物學活性的影響[D];華中科技大學;2009年

5 豐菁;RNAi沉默ADAMs家族基因對腫瘤細胞生物學活性的影響[D];華中科技大學;2009年

6 張璞;AT1RshRNA重組腺病毒載體在C6細胞中的功能表達及對SHR的AT1R分布的影響[D];山西醫(yī)科大學;2006年

7 王剛;冠狀動脈支架內再狹窄影像學評價及基因干擾防治機制研究[D];第四軍醫(yī)大學;2010年

8 周磊;MEK1和MEK2差異調節(jié)胰腺癌細胞功能的實驗研究[D];中國醫(yī)科大學;2010年

，

本文編號：2075261