携带siRNA的慢病毒载体抑制SHR阴茎海绵体平滑肌细胞S1PR3的表达

发布时间：2018-04-03 18:06

  本文选题：自发性高血压大鼠　切入点：1-磷酸鞘氨醇受体3　出处：《西南医科大学》2017年硕士论文

【摘要】：目的:筛选出能够高效特异性沉默自发性高血压大鼠(spontaneously hypertensive rats,SHR)阴茎海绵体平滑肌细胞1-磷酸鞘氨醇受体3(sphingosine-1-phosphate receptor 3,S1PR3)基因表达的siRNA慢病毒载体,并观察其对SHR阴茎海绵体平滑肌细胞ROCK1、ROCK2、eNOS表达的影响[1]。方法:以大鼠S1PR3基因mRNA序列作为干扰靶点,设计并合成3对靶向S1PR3的siRNA序列(siRNA1、siRNA2、siRNA3)及1对阴性对照序列,构建并包装成慢病毒载体[1]。体外培养SHR阴茎海绵体平滑肌细胞及魏-凯二氏大鼠(Wistar-Kyoto rats,WKY)阴茎海绵体平滑肌细胞,随机分为A组(SHR对照组)、B组(携带阴性对照病毒的SHR阴茎海绵体平滑肌细胞转染组)、C~E组(分别携带靶向S1PR3基因siRNA1~3号靶点慢病毒的SHR阴茎海绵体平滑肌细胞转染组)、F组(WKY对照组),以感染复数(MOI)=60转染SHR阴茎海绵体平滑肌细胞,转染72h后观察细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达情况,并用RT-PCR和Western blotting检测转染后细胞中S1PR3、ROCK1、ROCK2、eNOS mRNA及蛋白的表达情况[1]。结果:经基因测序证明合成的携带S1PR3基因siRNA 1~3号靶点的寡核苷酸链序列插入正确。荧光显微镜下观察发现携带靶向S1PR3的siRNA慢病毒载体及阴性对照病毒载体均能有效转染SHRCCSM细胞,且转染效率均80%[1]。C、D、E、F组的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达较A组下降(p0.05),其中E组抑制作用最为明显,使S1PR3基因mRNA及蛋白表达的抑制效率分别为(34.2±2.9)%、(77.7±4.7)%;ROCK1基因mRNA及蛋白表达的抑制效率分别为(33.3±1.4)%、(51.1±7.3)%;ROCK2基因mRNA及蛋白表达的抑制效率分别为(30.8±3.6)%、(58.32±5.5)%。S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达在A组与B组之间均无明显改变,A组eNOS基因mRNA及蛋白表达分别与B、C、D、E比较,无明显差别,但较F组显著降低(p0.01);与F组比较,E组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显差别,A、B、C、D组的S1PR3、ROCK1、ROCK2基因mRNA及蛋白表达高于F组(p0.05),A、B、C、D、E组eNOS基因mRNA及蛋白表达较F组显著降低(p0.01)[1]。结论:本研究构建的3条携带不同位点的S1PR3基因的siRNA慢病毒载体均能够显著抑制SHR阴茎海绵体平滑肌细胞S1PR3基因的表达,并能有效的抑制SHR阴茎海绵体平滑肌细胞中上调的RhoA/Rho激酶信号通路,其中携带siRNA3慢病毒载体的抑制效率最高[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鍙婅泲鐧借〃杈剧殑鎶戝埗鏁堢巼鍒嗗埆涓，

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