体外培养的豚鼠阴茎海绵体ICC表达nNOS的实验研究

目的 探索成年豚鼠阴茎海绵体中Cajal间质细胞(ICC)的分离、培养和鉴定方法以及神经型一氧化氮合酶(nNOS)的表达情况.方法 ①取豚鼠阴茎海绵体组织,采用酶消化法分离细胞,差速帖壁法纯化ICC,将细胞接种于DMEM培养基中培养,48 h后进行c-kit特异性抗体免疫荧光染色法鉴定ICC并用c-kit/nNOS特异性抗体免疫荧光双重染色法观察nNOS的表达情况.结果 培养24 h后细胞帖壁良好,倒置显微镜下显示ICC呈纺锤形,有两个或多个长的突起,48 h后细胞形态趋于稳定,免疫荧光染色可见ICC呈c-kit抗体染色阳性,c-kit/nNOS特异性抗体免疫荧光双重染色显示培养的ICC表达nNOS.结论 用混合酶消化法可以成功分离和培养出豚鼠阴茎海绵体ICC,豚鼠阴茎海绵体ICC可能参与一氧化氮(NO)的分泌和传导,在阴茎勃起中发挥重要作用.

Study on expression of nNOS in cultured ICC of guinea pig corpus cavernosum

Objective To explore the method for isolation, culture and identification of interstitial cells of Cajal （ICC） from corpus caver- nosum of guinea pigs and to observe the expression of nNOS in ICCs. Methods The corporal tissue was obtained from adult guinea pig. These ICCs were isolated by enzyme digestion and purified through differential attachment technique, then cell suspensions were inoculated onto culture dishes and cultured in DMEM medium. After 48 hours, these cells were identified by using c - kit of immunofluorescence staining and the expres- sion of nNOS in ICCs was observed by c - kit and nNOS immunofluoresccnce double staining. Results ICCs adhered well after cultured for 24 hours, and ICCs were spindle - shaped with two or more long processes under phase contrast microscopy. The morphological features of ICCs were stabilized, immunofluorescence staining showed these cultured cells were c - kit positive, and c - kit and nNOS double stai- ning showed these c - kit positive cells were nNOS positive. Conclusion Enzyme digestion method can successfully isolate and culture guinea pig penis ICCs. ICCs may participate in the secretion and conduction of NO in corpus cavernosum, and play a vital role in penile erection.