成人及家兔阴茎海绵体平滑肌细胞培养和鉴定

Culture and Identification of Human and Rabbit Corpus Cavernosum Smooth Muscle Cells

目的 :探索阴茎海绵体平滑肌细胞 (CCSM)体外培养及鉴定方法。 方法 :采用勃起功能正常的成年男子及新西兰家兔的新鲜阴茎标本 ,经处理后采用原代细胞培养法 ,对阴茎海绵体组织块进行培养 ,获得CCSM后用光镜、透射电镜和免疫组化等多种方法从细胞形态学及CCSM生长特性等不同侧面对培养的人及新西兰家兔的CCSM进行鉴定 ,并测定CCSM在体外培养时的细胞增殖情况。 结果 :经 2 4h潜伏期 ,体外培养的CCSM进入指数增长期 ,维持约 96h后进入平台期 ;从接种组织块至长成单层细胞约需 15～ 2 0d ,传代后约 4～ 7d后长成单层 ;传代后的细胞增殖旺盛 ,成束状生长 ,并呈现层叠排列及“峰 谷”现象 ;CCSM在体外可稳定传 7～ 12代 ,传代期间其形态及增殖活性变化不明显 ;各种检测鉴定均证实所获细胞为CCSM。 结论 :CCSM原代细胞培养法简便、稳定、可靠 ,对研究阴茎的勃起机制及勃起功能障碍的发生、发展、调控及其治疗药物筛选有重要理论及现实意义。

Objective: To investigate an effective method to produce large numbers of pure corporal smooth muscle cells in vitro according to the requirement of study. Methods: In this study, we used the primary tissue culture technique to isolate and culture the corpus cavernosum smooth muscle cells (CCSM) from human males with normal erectile function and New Zealand white rabbits. The cells were identified in regard to morphological and growing characteristics via immunohistochemical methods (including α-smooth muscle actin, desmin, myocin and factor Ⅷ related antigen), special dye techniques (including Masson and Van Gieson) and transmission electron microscope. Results: CCSM were isolated and cultured successfully with high purity. Morphologically, the cells were spindle shaped and grow on top of each other, resembling a 'hill and valley' in appearance. When characterized in immunohistochemistry, the cells were stained with α-smooth muscle actin, desmin and myocin, but not with anti-factor Ⅷ, an endothelial marker. Conclusion: The CCSM, which can be isolated and cultured successfully, may be used for further studying their biological function. The CCSM cultured in vitro was proved to be useful to evaluate and investigate the effect of some new medicine for penile erection. There is also a clinical and theoretical significance in further studying the experimental mechanisms of erectile dysfunction.