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

【摘要】： 一、編碼大鼠PRMT1基因shRNA質(zhì)粒的構(gòu)建和鑒定 目的:為研究蛋白精氨酸甲基轉(zhuǎn)移酶1(PRMT1)基因表達(dá)的改變與血管內(nèi)皮功能及動脈粥樣硬化的關(guān)系,我們構(gòu)建了PRMT1短發(fā)夾RNA質(zhì)粒(pPRMT1-shRNA)。 方法:在GenBank中查到大鼠PRMT1基因mRNA序列并輸入siRNA網(wǎng)上設(shè)計軟件OptiRNA中,選取兩條靶序列,設(shè)計并合成編碼shRNA序列的DNA單鏈,經(jīng)退火連接后,與雙酶切線性化處理回收的pGenesil-1質(zhì)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(zhì)粒后行酶切鑒定并測序。 結(jié)果:構(gòu)建的大鼠pPRMT1-shRNA經(jīng)酶切鑒定及測序與預(yù)期相符。 結(jié)論:本研究結(jié)果為進(jìn)一步進(jìn)行體外基因干擾PRMT1研究奠定了基礎(chǔ)。 二、PRMT1-shRNA質(zhì)粒轉(zhuǎn)染大鼠主動脈內(nèi)皮細(xì)胞條件的優(yōu)化 目的:優(yōu)化pPRMT1-shRNA在聚乙烯亞胺轉(zhuǎn)染劑jetPEITM-RGD介導(dǎo)下轉(zhuǎn)染原代培養(yǎng)大鼠主動脈內(nèi)皮細(xì)胞的轉(zhuǎn)染條件。 方法:貼塊法原代培養(yǎng)大鼠主動脈內(nèi)皮細(xì)胞,經(jīng)免疫細(xì)胞化學(xué)法(S-P法)鑒定,取3～4代細(xì)胞,按不同jetPEITM-RGD/pPRMT1- shRNA比例進(jìn)行轉(zhuǎn)染,24小時后測定各組轉(zhuǎn)染效率及細(xì)胞存活率。 結(jié)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(zhuǎn)染效率最高,為53.54%。 結(jié)論:本研究為高效地進(jìn)行細(xì)胞轉(zhuǎn)染及進(jìn)一步研究基因干擾PRMT1基因?qū)ρ獫{同型半胱氨酸(Hcy)、非對稱性二甲基精氨酸(ADMA)水平和血管內(nèi)皮功能的影響奠定了基礎(chǔ)。 三、PRMT1-shRNA質(zhì)粒對大鼠主動脈內(nèi)皮細(xì)胞PRMT1表達(dá)的影響 目的:探討pPRMT1-shRNA在體外對大鼠主動脈內(nèi)皮細(xì)胞PRMT1基因mRNA表達(dá)水平的影響。 方法:用pPRMT1-shRNA1、pPRMT1-shRNA2陽性質(zhì)粒、陰性對照pHK質(zhì)粒轉(zhuǎn)染大鼠主動脈內(nèi)皮細(xì)胞,同時設(shè)立空白對照組。轉(zhuǎn)染12、24小時后分別收集各組細(xì)胞,提取細(xì)胞總RNA,通過逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(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(dá)情況。 結(jié)果:pPRMT1-shRNA1及pPRMT1-shRNA2轉(zhuǎn)染組12、24小時PRMT1基因mRNA表達(dá)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(zhuǎn)染組24小時PRMT1基因mRNA表達(dá)受抑較12小時顯著(P0.05),且pPRMT1- shRNA1組比pPRMT1-shRNA2轉(zhuǎn)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(dá)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(jié)論:本研究所構(gòu)建的pPRMT1-shRNA1及pPRMT1-shRNA2可在體外高效特異地抑制PRMT1的表達(dá),為進(jìn)一步的體內(nèi)實驗奠定了基礎(chǔ)。
[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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R346;R541.4

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 王猛;ANXA2在視網(wǎng)膜新生血管形成過程中的表達(dá)及作用機(jī)制[D];青島大學(xué);2012年

相關(guān)碩士學(xué)位論文 前8條

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

2 徐廣芬;缺氧誘導(dǎo)因子-1α在皮膚鱗狀細(xì)胞癌中的表達(dá)及促進(jìn)細(xì)胞增殖作用[D];華中科技大學(xué);2011年

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

4 豐菁;RNAi沉默ADAMs家族基因?qū)δ[瘤細(xì)胞生物學(xué)活性的影響[D];華中科技大學(xué);2009年

5 豐菁;RNAi沉默ADAMs家族基因?qū)δ[瘤細(xì)胞生物學(xué)活性的影響[D];華中科技大學(xué);2009年

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

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

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

，

本文編號：2075261