小鼠膀胱平滑肌原代细胞体外培养及纤维连接蛋白1的小干扰RNA转染对周期、凋亡的影响  

Primary culture of mouse bladder smooth muscle cells in vitro and effects of fibronection 1-small interfering RNA transfection on cell cycle and apoptosis

作　　者：李鹏[1] 顾朝辉[1] 贾占奎[1] 杨锦建[1] Li Peng;Gu Chaohui;Jia Zhankui;Yang Jinjian(Department of Urology,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院泌尿外科,450052

出　　处：《中华实验外科杂志》2020年第8期1409-1411,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(81570685)。

摘　　要：目的建立一种体外连续传代培养小鼠膀胱平滑肌细胞(BSMCs)的方法并探讨纤维连接蛋白1(FN1)沉默对小鼠BSMCs周期分布和凋亡的影响。方法采取多酶联合消化法进行原代培养,分离获取小鼠原代BSMCs,通过形态学观察和免疫荧光法对细胞进行鉴定;使用细胞计数试剂盒(CCK-8)法对其增殖特性进行检测。使用针对FN1的小干扰RNA(FN1-siRNA)转染BSMCs,将体外培养的BSMCs分为空白组(未转染)、阴性对照组(转染阴性对照NC-siRNA)和干扰组(转染FN1-siRNA),流式细胞仪检测细胞周期分布和凋亡率。3组间以上指标的比较采用单因素方差分析,两两比较采用SNK-q检验。结果从小鼠中获得的BSMCs纯度可达97%,细胞呈典型的"峰-谷"样结构。免疫荧光鉴定可见胞质内α-平滑肌肌动蛋白(α-SMA)阳性表达。FN1-siRNA转染后[G0/G1(68.20±3.82)%、S(17.78±1.48)%、G2/M(14.04±2.04)%]与转染前[G0/G1(49.59±2.38)%、S(28.75±1.94)%、G2/M(21.71±1.93)%]比较,增殖水平降低;与转染前[(3.12±0.65)%]比较,转染后[(17.36±2.62)%]凋亡率升高(F=89.346,P<0.05)。结论体外培养获得的小鼠BSMCs具有较好增殖能力。靶向沉默FN1的表达可诱导BSMCs周期阻滞和凋亡。Objective To establish a method of mice bladder smooth muscle cells(BSMCs)culture by continuous subculture in vitro and discuss the effects of silencing Fibronection 1 on cycle distribution and apoptosis of mice BSMCs.Methods The primary BSMCs were cultured and isolated by multi-enzyme combined digestion method.The cells were identified by morphological observation and immunofluorescence,and the proliferation characteristics were detected by cell counting kit-8(CCK-8).In vitro cultured BSMCs were divided into blank group(untransfected),negative control group[transfected with negative control NC-small interfering RNA(siRNA)]and interference group(transfected with FN1-siRNA).The cell cycle distribution and apoptosis rate were detected by flow cytometry.Results The cells had a typical"peak-valley"structure.Immunofluorescent staining indicated the expression ofα-smooth muscle actin(α-SMA)in cytoplasm.The proliferation of FN1-siRNA transfected cells[G0/G1(68.20±3.82)%,S(17.78±1.48)%,G2/M(14.04±2.04)%]was inhibited as compared with that before transfection[G0/G1(49.59±2.38)%,S(28.75±1.94)%,G2/M(21.71±1.93)%].Compared with before transfection[(3.12±0.65)%],the apoptosis rate increased after transfection[(17.36±2.62)%,F=89.346,P<0.05].Conclusion Mice BSMCs cultured in vitro had a good proliferative ability.FN1-siRNA transfection could induce cell cycle arrest and apoptosis of BSMCs.

关 键 词：膀胱平滑肌细胞 原代培养 纤维连接蛋白1 细胞周期 脱噬作用 

分 类 号：R-33[医药卫生]