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目的探索培养老年勃起功能障碍(ED)大鼠阴茎海绵体平滑肌细胞(SMC s)生物学特性稳定、细胞纯度高、简单易行的一种新方法。方法电生理仪检测24月龄雄性大鼠阴茎海绵体压力和平均动脉压比值(ICP/MAP)来评价阴茎勃起功能,从而选择老年ED大鼠。分别采用组织块法、酶消化法和酶消化差速贴壁法分离培养24月龄ED大鼠SMCs;采用存活率、生长曲线、相差显微镜和HE染色等,比较不同方法培养SMCs的生物学特性;免疫组化、免疫荧光和流式细胞仪通过检测α-SM-actin、Myosin和Desmin的阳性率来鉴定SMCs,并比较不同方法培养SMCs的纯度。结果 ICP/MAP低于0.45入选老年ED大鼠组。细胞存活率提示酶消化法和组织块法存活率分别为93%和90%,酶消化差速贴壁法显著增加到99%(P<0.05);生长曲线提示酶消化差速贴壁法细胞数量在第5～7天也显著增加(P<0.05);酶消化差速贴壁法培养细胞在相差显微镜和HE染色下形态基本一致,呈梭形生长。免疫组化、免疫荧光和流式鉴定是SMCs,组织块法和酶消化法原代培养的SMCs纯度平均分别为45%和82%,酶消化差速贴壁法SMCs纯度显著增加到98%(P<0.05)。α-SM-act|in、Myosin和Desmin在SMCs中有大量表达,在11代以内SMCs纯度维持较高水平(95%以上)。结论酶消化差速贴壁法快速简便,可在一定范围内提高SMCs纯度;用此法培养的SMC s,生物学特性稳定,细胞纯度高,为建立SMC s细胞株奠定基础。
Objective To explore a new method to cultivate the stable biological characteristics, high cell purity and easy operation of corpus cavernosum smooth muscle cells (SMCs) in senile erectile dysfunction (ED) rats. Methods Erectile function was evaluated by electrophysiological examination of penile cavernous pressure and mean arterial pressure ratio (ICP / MAP) in 24-month-old male rats to select aged ED rats. The SMCs of 24-month-old ED rats were isolated and cultured by tissue block method, enzymatic digestion method and enzymatic digestion differential adhesion method. The biological characteristics of SMCs were compared by using different methods in terms of survival rate, growth curve, phase contrast microscope and HE staining Immunofluorescence, immunofluorescence and flow cytometry were used to identify SMCs by detecting the positive rate of α-SM-actin, Myosin and Desmin, and compared the purity of SMCs cultured by different methods. Results ICP / MAP was lower than 0.45 into the aged ED rats. Cell survival rate suggested that the enzymatic digestion and tissue block survival rates were 93% and 90%, respectively. The rate of enzymatic digestion was significantly increased to 99% (P <0.05). The growth curve suggested that enzymatic digestion differential adherent cells The number increased significantly from the 5th to 7th day (P <0.05). The morphology of the cultured cells differentiated by enzymatic digestion and adherent method was almost the same under the phase contrast microscope and HE staining. Immunohistochemistry, immunofluorescence and flow cytometry identified SMCs. The average purity of SMCs cultured by tissue block method and enzymatic digestion method were 45% and 82%, respectively. The purity of SMCs by differential enzyme digestion was significantly increased to 98% P <0.05). α-SM-act | in, Myosin and Desmin were abundantly expressed in SMCs. SMCs maintained high purity (over 95%) within the 11th generation. Conclusion The enzymatic digestion differential adherence method is rapid and simple, and can improve the purity of SMCs within a certain range. The SMC s cultured in this way has stable biological characteristics and high cell purity, which lays the foundation for the establishment of SMC s cell line.