Screening lentiviral vectors carrying effective siRNA of the ROCK2 gene / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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 x 10(4) 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.</p><p><b>RESULTS</b>The transfection efficiency of the SHR corpus cavernosum smooth muscle cells was > 50%. Compared with group A, the expression 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 significantly in E ([16.81 +/- 5.94]%, P < 0.05).</p><p><b>CONCLUSION</b>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.</p>