短发夹RNA对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型基因表达的抑制作用  

The inhibitive effect of shRNA on the phosphodiesterase type 5 in smooth muscle cells of rat corpus cavernosum

作　　者：李忠远[1] 刘继红[1] 封江南 肖恒军[1] 詹鹰[1] 张朝晖[1] 王涛[1] 王少刚[1] 叶章群[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030 [2]武汉晶赛生物工程技术有限公司

出　　处：《中华实验外科杂志》2009年第11期1521-1522,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30471736）

摘　　要：目的观察短发夹RNA（shRNA）对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型（PDE5）基因表达的抑制作用，探讨运用RNA干扰（RNAi）技术治疗勃起功能障碍（ED）的可行性。方法构建靶向大鼠PDE5基因的shRNA重组腺病毒rAd-rPDE5-shRNA，将其转染大鼠阴茎海绵体平滑肌细胞48h后，通过荧光标签进行显微计数确定转染效率，并以逆转录-聚合酶链反应（RTPCR）及Western blot检测PDE5基因的表达水平。结果rAd—rPDE5-shRNA构建成功，转染大鼠阴茎海绵体平滑肌细胞效率达95％以上，并使PDE5基因表达在mRNA水平抑制（80．78±2．30）％，在蛋白水平抑制（67．39±3．33）％。结论以腺病毒为载体表达的shRNA能稳定、有效地抑制大鼠阴茎海绵体平滑肌细胞PDE5基因的表达。Objective To down-regulate the expression of phosphodiesterase type 5 （PDE5） gene by short hairpin RNA （shRNA） in smooth muscle cells of rat corpus cavernosum, and investigate the feasibility of gene therapy for erectile dysfunction （ED）. Methods A recombinant adenovirus, rAd-rP- DE5-shRNA, targeting the PDE5 gene of rats was constructed. The rAd-rPDES-shRNA was transfected into the smooth muscle cells of rat corpus cavernosum. The efficiency of transfection was evaluated by cell counting of EGFP expressed cells under fluorescence microscopy, and the expression of PDE5 gene was detected by RT-PCR and Western blot at the 48th h post-transfection. Results rAd-rPDE5-shRNA was identified and transfected at a MOI of 30 pfu per cell for 48 h. The expression of PDE5 in the smooth muscle cells of rat corpus cavernosum was inhibited by （ 80.78 ± 2.30） % at mRNA level, and （ 67.39 ± 3.33） % at protein level. Conclusion The shRNA delivered by the recombinant adenovirus can stably and effectively inhibit the expression of PDE5 in smooth muscle cells of rat corpus cavernosum.

关 键 词：RNA干扰 阴茎海绵体 平滑肌细胞 磷酸二酯酶 基因表达 

分 类 号：R698.2[医药卫生—泌尿科学]