携带siRNA的慢病毒载体抑制自发性高血压大鼠阴茎海绵体平滑肌细胞S1PR3的表达

发布时间：2018-04-18 03:10

本文选题：自发性高血压大鼠 + -磷酸鞘氨醇受体　；参考：《中华男科学杂志》2017年02期

【摘要】：目的:筛选出能够高效特异性沉默自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞(CCSMC)内1-磷酸鞘氨醇受体3(S1PR3)基因表达的siRNA慢病毒载体,并观察其对SHR CCSMC ROCK1、ROCK2、e NOS表达的影响。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。体外培养SHR CCSMC及魏-凯二氏大鼠(WKY)CCSMC,随机分为A组(SHR对照组)、B组(携带阴性对照病毒的SHR CCSMC转染组)、C~E组(分别携带靶向S1PR3基因siRNA 1~3号靶点慢病毒的SHR CCSMC转染组),F组(WKY对照组),以感染复数(MOI)=60转染SHR CCSMC,转染后观察细胞绿色荧光蛋白(GFP)表达情况,并用RT-PCR和Western印迹检测转染后细胞中S1PR3、ROCK1、ROCK2、e NOS mRNA及蛋白的表达情况。结果:经基因测序证明慢病毒载体构建成功。荧光显微镜下观察B~E组细胞转染效率均80%。与A组相比,B组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),C、D、E、F组的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均较A组显著下降(P均0.05),其中E组抑制作用最为明显,使S1PR3 mRNA及蛋白表达的抑制效率分别为(34.2±2.9)%、(77.7±4.7)%;ROCK1 mRNA及蛋白表达的抑制效率分别为(33.3±1.4)%、(51.1±7.3)%;ROCK2 mRNA及蛋白表达的抑制效率分别为(30.8±3.6)%、(58.32±5.5)%。A组e NOS mRNA及蛋白表达分别与B、C、D、E比较无明显差异(P均0.05),但较F组显著降低(P0.05);与F组比较,E组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),A、B、C、D组的S1PR3、ROCK1、ROCK2 mRNA及蛋白表达均显著高于F组(P均0.05),A、B、C、D、E组e NOS mRNA及蛋白表达均较F组显著降低(P均0.05)。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 CCSMC内S1PR3基因的表达,并能有效抑制SHR CCSMC中上调的Rho A/Rho激酶信号通路,其中携带siRNA3慢病毒载体的抑制效率最高。
[Abstract]:Aim: to screen out the siRNA lentivirus vector which can specifically silence the gene expression of 1-sphingosine receptor 3nS1PR3 in the smooth muscle cells of the cavernous body of spontaneously hypertensive rats (SHR), and to observe the effect of the vector on the expression of SHR CCSMC rok1 roCK2e NOS.Methods: using the mRNA sequence of rat S1PR3 gene as the interference target, we designed and synthesized 3 pairs of siRNA sequences targeting S1PR3 and 1 pair of negative control sequences, and constructed and packaged the lentivirus vector.SHR CCSMC and WKYY CCSMCs were cultured in vitro. They were randomly divided into two groups: group A (SHR CCSMC transfection with negative control virus) group (SHR CCSMC transfection group with siRNA 1 ~ 3 target for S1PR3 gene 1 ~ 3) and control group B (SHR CCSMC transfection group with siRNA 1 ~ 3 target for S1PR3 gene 1 ~ 3) was randomly divided into two groups: group A and group B (SHR CCSMC transfection group with negative control virus) (SHR CCSMC transfection group with siRNA 1 ~ 3 target for S1PR3 gene).SHR CCSMCs were transfected with MMOION60 and the expression of green fluorescent protein (GFP) was observed after transfection.RT-PCR and Western blot were used to detect the expression of S1PR3, ROCK1, ROCK2e NOS mRNA and protein in transfected cells.Results: the construction of lentivirus vector was proved by gene sequencing.The transfection efficiency of BNE group was 80% under fluorescence microscope.Compared with group A, the expression of mRNA and protein in S1PR3, ROCK1, ROCK2 and protein in group S1PR3, ROCK1, ROCK2 and protein were significantly decreased in group S1PR3, ROCK1, ROCK2 and protein, especially in group E (P < 0. 05), and the inhibitory effect in group E was the most obvious, and the expression of mRNA and protein in group S1PR3, ROCK1, ROCK2 and protein were significantly decreased compared with group A (P < 0. 05).The inhibitory rates of S1PR3 mRNA and protein expression were 34.2 卤2.9 and 77.7 卤4.7, respectively. The inhibitory rates of S1PR3 mRNA and protein expression were 33.3 卤1.4 and 51.1 卤7.3, respectively. The inhibition rates of S1PR3 mRNA and protein expression were 30.8 卤3.60.32 卤5.55.50.There was no significant difference between group A and group A in NOS mRNA and protein expression (P 0.05).However, compared with F group, the expression of S1PR3, ROCK1, ROCK2 mRNA and protein in E group were not significantly changed compared with that in F group, and the expression of S1PR3, ROCK1, ROCK2 mRNA and protein in group S1PR3, ROCK1, ROCK2 and protein were significantly higher than those in group F, and the expression of e NOS mRNA and protein in group E were significantly lower than those in group F (P < 0.05).Conclusion: the three siRNA lentivirus vectors with different S1PR3 loci can significantly inhibit the expression of S1PR3 gene in SHR CCSMC and the up-regulated Rho A/Rho kinase signaling pathway in SHR CCSMC.Among them, the inhibition efficiency of siRNA3 lentivirus vector was the highest.
【作者单位】：西南医科大学附属医院泌尿外科;
【基金】：四川省科技厅基金(14JC0803) 四川省教育厅基金(15ZA0164) 四川省人力资源和社会保障厅回国人员科研基金(川人社办发[2016]64号)~~
【分类号】：R544.1;R698

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