猪冠状动脉平滑肌细胞的分离、培养及鉴定

目的:建立体外分离培养猪冠状动脉血平滑肌细胞(PCASMC)的技术方法并观察其生长特征.方法:酶联合消化法原代分离猪冠状动脉来源的CASMC,利用倒置显微镜观察细胞生长情况;免疫荧光染色法鉴定细胞;台盼蓝法、绘制生长曲线法测定冠状动脉PCASMC传代细胞的成活率和生长特征.结果:分离培养的细胞3d后呈梭形生长,7~8d后生长迅速可传代;第2代细胞在显微镜下观察3~5d即可见典型"峰-谷"状生长;免疫荧光染色显示胞浆内α-平滑肌肌动蛋白表达阳性,细胞成活率为97.5%;细胞生长曲线近似"S"形.结论:本研究建立了高效分离和培养PCASMC的方法,细胞均稳定地表达α-平滑肌肌动蛋白,为血管疾病的研究提供了理想的细胞模型.

Isolated Culture and Identification of Mouse Aortic Vascular Smooth Muscle Cell

Objective:To build the technical method of isolated culture porcine coronary artery smooth muscle cell(PCASMC)in vitro and observe their growth characteristics.Methods:PCASMC of original generation separation from coronary artery in porcine by enzymatic digestion method were prepared and the conditions of cellular growth were observed under inverted microscope.The PCASMC were identified by immunofluorescence staining.The survival ratio,growth and proliferation characteristics of passage PCASMC were measured by trypan blue method and growth curve method.Result:After cells were cultured for three days with enzyme digestion method,the cells were spindle growth and rapidly growth until 7-8days and could be passaged.The second generation cells had a typical "peak-valley" like growth under microscope.The expressions of intracellularα-smooth muscle actin in cells were positive by immunofluorescence stain,cell survival rate was 96.5%.Cells growth curve were similar to "S" shape.Conclusion:This study provided efficient isolation methods to culture porcine artery PCASMC,which stably expressα-smooth porcine actin.It may be provide an ideal cellular model for the research of coronary artery diseases.