酶消化差速贴壁法分离培养老年ED大鼠阴茎海绵体平滑肌细胞的研究

Isolating smooth muscle cells from corporal cavernosum of aging rats with erectile dysfunction using enzyme digestion velocity sedimentation method

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 assess culture methods for isolation of smooth muscle cells （SMCs） from corporal cavernosum of aging rats with ED. Methods Twenty four-month-old ED rats with ICP/MAP less than 0.45 were used in the study. SMCs were isolated by tissue culture, enzyme digestion and enzyme digestion velocity sedimentationmethods respectively. The cell survival rate, cell growth curve, cell morphology were used to evaluate biological characteristics, and the isolated SMCs were identified by the expressions of a -SM-actin, myosin and desmin. Results The isolated SMCs appeared spindle morphologically under phase contrast microscope. The cell livability and growth curve increased using enzyme digestion velocity sedimentation method （P〈0.05）. The purity of isolated SMCs using tissue culture and enzyme digestion methods was 45% and 82% respectively, whereas it was98% using enzyme digestion velocity sedimentation method （P〈0.05）. The purity of cells was high （〉95%） within the 11th passage. Conclusion The enzyme digestion velocity sedimentation method is convenient and feasible for isolation of SMCs with stable biological characteristics, which will benefit the establishement of cell strains.