携带ROCK2基因siRNA有效片段的慢病毒载体的筛选

目的:筛选能够显著抑制自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞内ROCK2基因表达的携带ROCK2基因siRNA的慢病毒载体。方法:设计并合成4个靶向ROCK2的siRNA片段,构建并包装成慢病毒载体。随机将5只SHR阴茎海绵体平滑肌细胞分为6组,每组每个样本3×104个细胞,每组5个样本,分别为:A组(未转染对照组)、B组(携带慢病毒转染组)、C~F组(分别携带靶向ROCK2基因siRNA 1~4号靶点的慢病毒转染组),以感染复(MOI)=80转染SHR阴茎海绵体平滑肌细胞,转染后48 h荧光显微镜下观察细胞GFP表达情况,并用RT-PCR检测各组被转染细胞ROCK2 mRNA的表达。结果:荧光显微镜下观察各组细胞转染效率均50%。与A组相比,B组ROCK2 mRNA的表达无明显改变(P﹥0.05);C、D、F组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组极显著下降(P0.01),抑制效率分别达到(43.91±8.19)%、(47.15±6.64)%、(25.7±6.03)%;E组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组显著下降(P0.05),抑制效率为(16.81±5.94)%。结论:本研究构建的4种携带ROCK2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内ROCK2基因的表达,其中有1种慢病毒载体抑制作用最强。

Screening lentiviral vectors carrying effective siRNA of the ROCK2 gene

Objective： To screen the lentiviral vector carrying siRNA and capable of significantly suppressing the ROCK2 gene expression in the corpus cavernosum smooth muscle cells of spontaneously hypertensive rats （SHR）. Methods： We designed and synthesized 4 siRNA fragments targeting the ROCK2 gene and packaged them into lentiviral vectors. We collected corpus cavernosum smooth muscle cell samples from 5 male SHRs and randomly divided them into groups A （ non-transfection control） , B （ GFP lentiviral transfection）, C, D, E, and F （lentiviral transfection with siRNA fragments 1 -4 targeting the ROCK2 gene）. Each group consisted of 5 samples and each sample 3 × 104 cells. At 48 hours after transfecting MOI = 80 into the SHR corpus cavernosum smooth muscle cells, we detected the expression of GFP under the fluorescent microscope and the mRNA expression of the ROCK2 gene by RT-PCR. Results ： The transfection efficiency of the SHR corpus cavernosum smooth muscle cells was 〉 50%. Compared with group A, the ex- pression of ROCK2 mRNA in the corpus cavernosum smooth muscle cells showed no remarkable change in group B （P 〉 0.05 ） but was inhibited very significantly in C （ 43.91 ±8.19 ] % ）, D （ 47.15± 6.64 ] % ）, and F （ 25.7 ±6.03 ] % ） （ P 〈 0.01 ）, and signi- ficantly in E （ 16.81 ±5.94 ] %, P 〈 0.05）. Conclusion ： We successfully constructed 4 lentiviral vectors carrying siRNA targeting the ROCK2 gene, all of which can significantly suppress the ROCK2 expression in the SHR corpus cavernosum smooth muscle cells, and one has a highly strong inhibitory effect. NatlJAndrol, 2014, 20 （5） ： 392 -399