短发夹RNA靶向沉默大鼠阴茎海绵体平滑肌细胞胰岛素样生长因子结合蛋白-3的研究  

Short hairpin RNA silencing the expression of insulin-like growth factor binding protein 3 in corpus cavernosal smooth muscle cell in rat

作　　者：蒲小勇[1] 周香雪[2] 肖恒军[3] 林楚琪 彭伟华[1] 毕学成[1] 李东[1] 刘久敏[1] 

机构地区：[1]广东省医学科学院广东省人民医院泌尿外科,广州510080 [2]中山大学附属第一医院东院神经内科,广州510700 [3]中山大学附属第三医院泌尿外科,广州510600

出　　处：《中华实验外科杂志》2017年第8期1367-1370,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金项目（81570691、81270855）;广州市科技计划项目（2014J4100041）;广东省中医药局科研项目（20161001）;广东省人民医院科研项目（Y012012012）

摘　　要：目的 筛选靶向抑制大鼠阴茎海绵体平滑肌细胞（CCSMC）中胰岛素样生长因子结合蛋白-3（IGFBP-3）基因表达最有效的短发夹RNA（shRNA）,为基因沉默IGFBP-3治疗大鼠勃起功能障碍提供依据.方法 构建4对靶向IGFBP-3的短发夹RNA（shRNA）质粒和阴性对照,体外原代培养CCSMC,分6组：空白对照组、转染pGPU6/GFP/Neo-IGFBP-3 （611）组、转染pGPU6/GFP/Neo-shIGFBP-3 （526）组、转染pGPU6/GFP/Neo-shIGFBP-3 （866）组、转染pGPU6/GFP/Neo-shIGFBP-3 （710）组和转染pGPU6/GFP/Neo-shNC阴性对照组;转染48h后各组分别用实时定量聚合酶链反应（PCR）和Western blot方法检测靶向沉默IGFBP-3的效果.结果 CCSMC转染后实时定量PCR和Western blot检测发现,pGPU6/GFP/Neo-shIGFBP3-526、pGPU6/GFP/Neo-shIGFBP3-611、pGPU6/GFP/Neo-shIGFBP3-866均有明显的沉默效果,与空白对照组和转染pGPU6/GFP/Neo-shNC组比较差异有统计学意义,而其中pGPU6/GFP/Neo-shIGFBP3-526沉默效果最明显,实时定量PCR和Western blot结果显示分别可以抑制72%和79%.结论 靶向IGFBP-3基因的shRNA能够高效抑制大鼠阴茎海绵体平滑肌细胞中靶基因的表达,为进一步体内实验奠定基础.Objective To screen the most effective short hairpin RNA （shRNA） which down-regulate the insulin-like growth factor binding protein 3 （IGFBP-3） in corpus cavernosal smooth muscle cell （CCSMC） in vitro.Methods 4 pair IGFBP-3 small interfering RNA （siRNA）,and plasmid constructs for U6 promoter plasmid vector （pGPU6/GFP/Neo-IGFBP-3-shRNA） and negative control vector were constructed.Corpus cavernosal smooth muscle cells （CCSMC） were primary cultured and divided into 6 groups： the CCSMCs-only,transfection pGPU6/GFP/Neo-IGFBP-3 （611） group,transfection pGPU6/GFP/Neo-IGFBP-3 （526） group,transfection pGPU6/GFP/Neo-IGFBP-3 （866） group,transfection pGPU6/GFP/Neo-IGFBP-3 （710） group and the negative control group （transfection with pGPU6/GFP/Neo-shNC）.The silencing effection was determined by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting.Results CCSMCs transfected with shRNA showed an obviously decrease of IGFBP-3 in mRNA and protein levels compared with CCSMCs transfected with negative control vector and the CCSMCs-only in transfection pGPU6/GFP/Neo-IGFBP-3 （611） group,pGPU6/GFP/Neo-IGFBP-3 （526） group and pGPU6/GFP/Neo-IGFBP-3 （866） group.The most inhibiting effective were observed in trasnfection pGPU6/GFP/Neo-shIGFBP3-526 group.The 72% and 79% inhibiting effection were determined in real-time PCR and Western blotting.ConclusionshRNA can obviously silence the expression of IGFBP-3 in CCSMCs.It may be an effective methods to improved erection in vivo.

关 键 词：勃起功能障碍 胰岛素样生长因子结合蛋白-3 基因治疗 RNA干扰技术 大鼠 

分 类 号：R698[医药卫生—泌尿科学]