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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(P<0.05);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。
OBJECTIVE: To screen siRNA lentiviral vectors capable of efficiently and specifically silencing silencing of sphingosine-1-phosphate 3-phosphate (SIPPR) gene in penile smooth muscle cells (SCSMCs) of spontaneously hypertensive rats (SHRs) On SHR CCSMC ROCK1, ROCK2, e NOS expression. Methods: Three S1PR3 targeting siRNA sequences (siRNA1, siRNA2, siRNA3) and a pair of negative control sequences were designed and synthesized based on the mRNA sequence of S1PR3 gene in rat and constructed and packaged into lentiviral vector. SHR CCSMC and WKY CCSMC were cultured in vitro and divided into group A (SHR control group), group B (SHR CCSMC group carrying negative control virus) and group C to E SHR CCSMC transfection group targeting S1PR3 siRNA target 1 to target 3 lentivirus) and F group (WKY control group) were transfected with SHR CCSMC at a multiplicity of infection (MOI) = 60, and the expression of green fluorescent protein (GFP). The expression of S1PR3, ROCK1, ROCK2, e NOS mRNA and protein in transfected cells were detected by RT-PCR and Western blot. Results: The lentiviral vector was successfully constructed by gene sequencing. Under the fluorescence microscope, the transfection efficiency of B ~ E group cells were> 80%. Compared with group A, the expressions of S1PR3, ROCK1 and ROCK2 mRNA and protein in group B were not significantly changed (all P> 0.05), and the expressions of S1PR3, ROCK1 and ROCK2 mRNA and protein in group C, D, E and F The inhibition of S1PR3 mRNA and protein expression was (34.2 ± 2.9)% and (77.7 ± 4.7)% respectively in group A (all P <0.05), and the inhibitory effect of group E was the most obvious. The expression of ROCK1 mRNA and protein (33.3 ± 1.4)% and (51.1 ± 7.3)%, respectively. The inhibitory rates of ROCK2 mRNA and protein expression were (30.8 ± 3.6)% and (58.32 ± 5.5)%, respectively. There was no significant difference in eNOS mRNA and protein expression between group A and B (P> 0.05), but significantly lower than group F (P <0.05). Compared with group F, the expression of S1PR3, ROCK1 (P> 0.05). The mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 in groups A, B, C and D were significantly higher than those in group F (P <0.05), while A The expression of eNOS mRNA and protein in group B, C, D and E were significantly lower than those in group F (all P <0.05). CONCLUSION: The siRNA lentiviral vector carrying three siRNAs with different sites of S1PR3 constructed in this study can significantly inhibit the expression of S1PR3 gene in SHR CCSMC and inhibit Rho A / Rho kinase signaling pathway up-regulated in SHR CCSMC effectively The siRNA3 lentiviral vector has the highest inhibition efficiency.