腺病毒载体介导PDE5-shRNAs对大鼠阴茎海绵体平滑肌细胞游离钙的影响  

Impact of Adenovirus-Mediated PDE5-shRNAs on the Free Calcium Level in the Smooth Muscle Cells of Rat Corpus Cavernosum

作　　者：潘运高[1] 刘继红[1] 詹鹰[1] 王涛[1] 万志华[1] 李忠远[1] 刘云[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,湖北武汉430030

出　　处：《中华男科学杂志》2009年第10期882-885,共4页National Journal of Andrology

基　　金：国家自然科学基金(30471736)~~

摘　　要：目的:观察腺病毒载体介导的PDE5-shRNAs对大鼠阴茎海绵体平滑肌细胞游离钙的影响,探讨利用RNAi技术治疗勃起功能障碍(ED)的可行性。方法:利用构建的携带2条靶向PDE5 mRNA靶位点的shRNAs重组腺病毒转染大鼠阴茎海绵体平滑肌细胞,同时设立阴性对照组和空白对照组,于转染后24、48、72 h用钙离子荧光探针Fluo-3/AM进行染色,然后在激光扫描共聚焦显微镜下动态检测细胞内钙离子荧光强度,以平均荧光指数(FI)反映细胞内游离钙的相对水平。结果:实验组在转染PDE5-shRNAs重组腺病毒24、48、72 h后钙离子荧光指数分别为829.3±7.8、801.5±9.5、856.3±8.7,均明显低于阴性对照组的1106.3±10.8、1121.3±10.2、1058.5±12.1和空白对照组的1076.6±9.7、1133.4±11.2、1104.3±10.5,差异有统计学意义(P<0.05)。结论:腺病毒介导的靶向PDE5基因的shRNAs能够明显降低大鼠阴茎海绵体平滑肌细胞内游离钙水平。Objective： To observe the influence of recombinant adenovirus-mediated PDE5-shRNAs on the free calcium level in rat penile smooth muscle cells,and to explore the feasibility of gene therapy for erectile dysfunction（ED）.Methods： Smooth muscle cells of the rat corpus cavernosum were transfected with constructed rAd-rPDE5-shRNAs and then dyed with the calcium fluorescent probe Fluo-3/AM at 24,48 and 72 hours.The dynamic changes of the calcium fluorescence intensity were observed under the laser scanning confocal microscope（LSCM）.The relative level of intracellular calcium was determined by fluorescence indexes.Results： The fluorescence indexes of calcium at 24,48 and 72 hours were 829.3±7.8,801.5±9.5 and 856.3±8.7 in the rAd-rPDE5-shRNAs group,significantly lower than in the rAd-mock（1 106.3±10.8,1 121.3±10.2 and 1 058.5±12.1） and blank control group（1 076.6±9.7,1 133.4±11.2 and 1 104.3±10.5）（P0.05）.Conclusion： Adenovirus mediated shRNAs of the target PDE5 gene can significantly decrease the intracellular calcium level in the smooth muscle cells of the rat corpus cavernosum.

关 键 词：勃起功能障碍 基因治疗 磷酸二酯酶5型 重组腺病毒 细胞内钙 短发夹RNA 

分 类 号：R698.1[医药卫生—泌尿科学]