本文選題：自發(fā)性高血壓大鼠　切入點(diǎn)：1-磷酸鞘氨醇受體3　出處：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:篩選出能夠高效特異性沉默自發(fā)性高血壓大鼠(spontaneously hypertensive rats,SHR)陰莖海綿體平滑肌細(xì)胞1-磷酸鞘氨醇受體3(sphingosine-1-phosphate receptor 3,S1PR3)基因表達(dá)的siRNA慢病毒載體,并觀察其對SHR陰莖海綿體平滑肌細(xì)胞ROCK1、ROCK2、eNOS表達(dá)的影響[1]。方法:以大鼠S1PR3基因mRNA序列作為干擾靶點(diǎn),設(shè)計(jì)并合成3對靶向S1PR3的siRNA序列(siRNA1、siRNA2、siRNA3)及1對陰性對照序列,構(gòu)建并包裝成慢病毒載體[1]。體外培養(yǎng)SHR陰莖海綿體平滑肌細(xì)胞及魏-凱二氏大鼠(Wistar-Kyoto rats,WKY)陰莖海綿體平滑肌細(xì)胞,隨機(jī)分為A組(SHR對照組)、B組(攜帶陰性對照病毒的SHR陰莖海綿體平滑肌細(xì)胞轉(zhuǎn)染組)、C~E組(分別攜帶靶向S1PR3基因siRNA1~3號靶點(diǎn)慢病毒的SHR陰莖海綿體平滑肌細(xì)胞轉(zhuǎn)染組)、F組(WKY對照組),以感染復(fù)數(shù)(MOI)=60轉(zhuǎn)染SHR陰莖海綿體平滑肌細(xì)胞,轉(zhuǎn)染72h后觀察細(xì)胞綠色熒光蛋白(green fluorescent protein,GFP)的表達(dá)情況,并用RT-PCR和Western blotting檢測轉(zhuǎn)染后細(xì)胞中S1PR3、ROCK1、ROCK2、eNOS mRNA及蛋白的表達(dá)情況[1]。結(jié)果:經(jīng)基因測序證明合成的攜帶S1PR3基因siRNA 1~3號靶點(diǎn)的寡核苷酸鏈序列插入正確。熒光顯微鏡下觀察發(fā)現(xiàn)攜帶靶向S1PR3的siRNA慢病毒載體及陰性對照病毒載體均能有效轉(zhuǎn)染SHRCCSM細(xì)胞,且轉(zhuǎn)染效率均80%[1]。C、D、E、F組的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達(dá)較A組下降(p0.05),其中E組抑制作用最為明顯,使S1PR3基因mRNA及蛋白表達(dá)的抑制效率分別為(34.2±2.9)%、(77.7±4.7)%;ROCK1基因mRNA及蛋白表達(dá)的抑制效率分別為(33.3±1.4)%、(51.1±7.3)%;ROCK2基因mRNA及蛋白表達(dá)的抑制效率分別為(30.8±3.6)%、(58.32±5.5)%。S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達(dá)在A組與B組之間均無明顯改變,A組eNOS基因mRNA及蛋白表達(dá)分別與B、C、D、E比較,無明顯差別,但較F組顯著降低(p0.01);與F組比較,E組S1PR3、ROCK1、ROCK2 mRNA及蛋白的表達(dá)均無明顯差別,A、B、C、D組的S1PR3、ROCK1、ROCK2基因mRNA及蛋白表達(dá)高于F組(p0.05),A、B、C、D、E組eNOS基因mRNA及蛋白表達(dá)較F組顯著降低(p0.01)[1]。結(jié)論:本研究構(gòu)建的3條攜帶不同位點(diǎn)的S1PR3基因的siRNA慢病毒載體均能夠顯著抑制SHR陰莖海綿體平滑肌細(xì)胞S1PR3基因的表達(dá),并能有效的抑制SHR陰莖海綿體平滑肌細(xì)胞中上調(diào)的RhoA/Rho激酶信號通路,其中攜帶siRNA3慢病毒載體的抑制效率最高[1]。
[Abstract]:Objective: to screen out the efficient silencing of spontaneously hypertensive rats (spontaneously hypertensive, rats, SHR) corpus cavernosum smooth muscle cells of sphingosine 1-phosphate receptor (sphingosine-1-phosphate 1- 3 receptor 3, S1PR3) of the lentiviral vector of siRNA gene expression, and to observe the ROCK2 of SHR ROCK1, corpus cavernosum smooth muscle cells, the expression of eNOS: [1]. method. The rat S1PR3 gene mRNA sequence as target, designed and synthesized 3 pairs of siRNA targeting sequence of S1PR3 (siRNA1, siRNA2, siRNA3) and 1 negative control sequences, constructed and packaged into lentivirus vector [1]. in vitro SHR penile corpus cavernosum smooth muscle cells and Wei - Kay's rats (two Wistar-Kyoto rats. WKY) corpus cavernosum smooth muscle cells, were randomly divided into A group (SHR group), B group (SHR corpus cavernosum smooth muscle cells in transfection group carrying negative control virus group (C~E), respectively, articles SHR corpus cavernosum smooth muscle cells in transfection group with S1PR3 gene targeted siRNA1~3 targeting lentivirus), F group (WKY group), with the multiplicity of infection (MOI) in =60 transfected SHR cells was observed in corpus cavernosum smooth muscle cells transfected with green fluorescent protein 72h (green fluorescent protein, GFP) expression, and RT-PCR and Western blotting detection of transfected cells in S1PR3, ROCK1, ROCK2, eNOS, mRNA and the expression of [1]. protein by gene sequencing proved that the synthesis of oligonucleotides carrying S1PR3 gene sequence of siRNA 1~3 target. Insert the correct found with targeting siRNA lentiviral vector and negative S1PR3 virus vector can effectively control transfection of SHRCCSM cells under the fluorescence microscope, and the transfection efficiency were 80%[1].C, D, E, F and S1PR3 in group ROCK1, the expression of ROCK2 protein and mRNA decreased compared with the A group (P0.05), the inhibition of E group is the most obvious. S1PR3鍩哄洜mRNA鍙?qiáng)铔嬬櫧琛ㄨ緞勬姂鍒舵晥鐜囧垎鍒福?

本文編號：1706317