腺病毒介导的激活大鼠阴茎海绵体平滑肌细胞中NO/cGMP通路的实验研究  

Study of adenovirus-mediated saRNA activating NO/cGMP pathway in rat corpus cavernosum smooth muscle cells

作　　者：袁慧星[1] 王涛[1] 詹鹰[1] 王少刚[1] 刘继红[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

出　　处：《临床泌尿外科杂志》2013年第8期616-620,628,共6页Journal of Clinical Urology

基　　金：国家自然科学基金(编号30872574)

摘　　要：目的:探讨靶向大鼠iNOS基因的shRNA重组腺病毒载体对大鼠阴茎海绵体平滑肌细胞iNOS基因的激活作用,为阴茎勃起功能障碍(ED)的基因治疗提供实验依据。方法:将前期构建的重组腺病毒Ad5-iNOSrshRNA-EGFP(AdU6/shiNOS)和对照病毒AdU6/shControl,分别转染大鼠阴茎海绵体平滑肌细胞,分别在不同病毒MOI(25,50,75)值下72小时后采样检测。采用realtime RT-PCR半定量检测AdU6/shiNOS对细胞iNOS基因mRNA表达影响;Western-blot法检测海绵体平滑肌细胞iNOS蛋白表达变化。然后培养基中加LArg(10mmol/L),用酶联免疫法检测病毒转染72小时后海绵体平滑肌细胞内cGMP的浓度变化,记录AdU6/shiNOS对平滑肌细胞内cGMP的影响。结果:AdU6/shiNOS转染大鼠阴茎海绵体平滑肌细胞72小时后,和空白对照组、阴性对照组相比iNOS基因在mRNA和蛋白表达水平均显著上调(P<0.05),呈剂量依赖性,MOI=75时RNAa效果最好。而且转染72小时后,实验组原代平滑肌细胞内cGMP水平显著高于对照组及空白组(P<0.05)。结论:利用腺病毒介导的RNAa技术,提高海绵体平滑肌细胞iNOS基因表达获得成功,可以增加阴茎海绵体平滑肌细胞cGMP水平,激活了NO/cGMP通路,这为勃起功能障碍的基因治疗研究开辟了新的方向。Objective： To explore the activation of recombinant adenovirus expressing short hairpin RNA of rat inducible nitric oxide synthase to iNOS gene in rat corpus cavernosum smooth muscle cells in order to provide ex- perimental evidence for erectile dysfunction gene therapy. Methods： The pre-constructed adenovirus Ad5-iNOS- shRNA-EGFP（AdU6/shiNOS） and the control virus AdU6/shControl transfected into rat cavernous smooth mus- cle cells. Cells were examined for iNOS expression by Real-time quantitative PCR and western blotting and differ- ent MOI（25, 50, 75） after 72 hours. Cells were then treated with 10 mmol/L L-arginine. Cyclic guanosine mono- phosphate（cGMP） with enzyme immunoassay （EIA） system at 72 hours after transfecting was analyzed and the effect of AdU6/shiNOS on the cGMP of smooth muscle cell was recorded. Results： Compared with mock group and control group, AdU6/shiNOS transfected rat corpus cavernosum smooth muscle cells after 72 hours, iNOS gene expression at the mRNA and protein were significantly increased in a dose-dependent（P^0.05）, MOI： 75 works best for RNAa. Corpus cavernosum smooth muscle cells transfected with AdU6/shiNOS showed an in- crease of cGMP level compared with corpus cavernosum smooth muscle cells transfected with control vector and the mock group（P^0.05）. Conclusion： Success using of adenovirus-mediated RNAa technology to improve the iNOS gene expression in corpus cavernosum smooth muscle cells can increase the cGMP level in corpus cavernosum smooth muscle cell, and activative the NO / cGMP pathway, which opened a new research direction for the gene treatment of erectile dysfunction.

关 键 词：勃起功能障碍 基因治疗 RNA激活 INOS基因 大鼠 

分 类 号：R697[医药卫生—泌尿科学]