应用RNA干扰技术抑制PDE_5A_3基因在人阴茎海绵体平滑肌细胞表达

Inhibition of the PDE5A3 gene expression by RNA interference in smooth muscle cells of human corpus cavernosum

目的应用RNA干扰(RNAi)技术抑制人阴茎海绵体平滑肌细胞PDEsA3基因的表达,探讨应用该技术治疗勃起功能障碍(ED)的可行性。方法构建6个靶向人PDEsA3基因的短发夹RNA(shRNA)重组质粒,转染人阴茎海绵体平滑肌细胞48 h后,逆转录-聚合酶链反应(RT- PCR)及Western blot检测PDE5A3基因的表达抑制效果。结果抑制率最高的1、2、4号重组质粒使PDE5A3基因表达在mRNA水平分别抑制(66．26±4．02)％,(54．90±3．06)％,(23．83± 3．61)％;在蛋白质水平分别抑制(64．14±3．32)％,(49．21±2．96)％,(29．85±4．91)％。结论 RNAi能明显抑制人阴茎海绵体平滑肌细胞PDE5A3基因的表达,且抑制率具有序列相关性,是潜在的ED基因治疗新方法。

Objective To inhibit the expression of PDE5A3 gene by RNA interference （RNAi） in the smooth muscle cells of human corpus cavernosum and to investigate the feasibility of gene therapy for erectile dysfunction （ED）. Methods Six recombinant plasmids with shRNAs targeting the PDE5A3 gene of Homo sapien were constructed. The recombinant plasmids were transfected into smooth muscle cells of human corpus cavernosum. Inhibition of PDE5A3 gene was detected by RT-PCR and Western blot after 48 h. Results PDE5A3 gene was inhibited by （66.26 ± 4.02） %, （54.90 ± 3.06） 96 and （23.83 ± ,3.61 ） 96 at mRNA level and （64. 14 ± 3.32） 96, （49.21 ± 2.96） %, （29. 85 ± 4.91） 96 at protein level respectively by No. 1, 2 and 4 recombinant plasmids. Conclusion RNAi can inhibit the expression of PDESA3 gene and has the feature of sequences correlation of shRNA in the smooth muscle cells of human corpus cavernosum. RNAi technology is a possible new approach for gene therapy of ED.