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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 investigate the activation of iNOS gene in rat corpus cavernosum smooth muscle cells by shRNA recombinant adenovirus vector targeting rat iNOS gene and to provide experimental evidence for gene therapy of erectile dysfunction (ED). Methods: The recombinant adenovirus Ad5-iNOSrshRNA-EGFP (AdU6 / shiNOS) and the control virus AdU6 / shControl were transfected into rat corpus cavernosum smooth muscle cells at different MOI (25, 50, 75) 72 hours after the sampling test. Real-time RT-PCR was used to detect the effect of AdU6 / shiNOS on the mRNA expression of iNOS. The expression of iNOS protein was detected by Western-blot. Then, LArg (10mmol / L) was added to the medium. The concentration of cGMP in the corpus cavernosum smooth muscle cells was detected by enzyme-linked immunosorbent assay 72 hours after transfection. The effect of AdU6 / shiNOS on cGMP in smooth muscle cells was recorded. Results: Compared with the blank control group and the negative control group, the expression of iNOS mRNA and protein in AdU6 / shiNOS transfected rat corpus cavernosal smooth muscle cells were significantly increased at 72 hours (P <0.05) in a dose-dependent manner, RNAa works best at MOI = 75. After 72 hours of transfection, the level of cGMP in primary smooth muscle cells in experimental group was significantly higher than that in control group and blank group (P <0.05). CONCLUSION: The successful use of adenovirus-mediated RNAi to improve the expression of iNOS gene in sponge smooth muscle cells can increase cGMP level and activate NO / cGMP pathway in corpus cavernosum smooth muscle cells, which opens up the possibility of gene therapy for erectile dysfunction New direction