组织块消化法原代培养大鼠输精管平滑肌细胞及鉴定

Tissue digestion method for isolation and characterization of primary cultures of rat vas deferens smooth muscle cells

目的建立一种稳定高效、存活率高的大鼠输精管平滑肌细胞体外分离及培养方法,为相关研究提供理想的细胞模型。方法采用组织块消化法对大鼠离体输精管平滑肌细胞进行原代培养。采用形态学观察、HE染色、抗α-SMA免疫细胞化学荧光染色对细胞进行鉴定,台盼蓝染色计算细胞存活率及细胞总数,胰酶消化传代并绘制生长曲线。结果对照实验表明胶原酶Ⅱ消化效果优于胶原酶Ⅰ,最佳消化时间为60 min,同时控制吹打次数、沉淀时间及培养环境,获得了理想的平滑肌细胞总数及存活率。平滑肌细胞呈典型的梭形、长条形,可传代,且出现平滑肌细胞培养典型的"峰-谷"状特征,生长曲线为"S"型。经抗α-SMA免疫荧光鉴定,培养细胞为平滑肌细胞,纯度为(92.6±4.3)%。结论采用组织块消化法成功建立了大鼠输精管平滑肌细胞的体外分离及培养方法。

Objective To establish a stable and efficient method for isolation and culture of rat vas deferens smooth muscle cells（VDSMCs）,and lay an ideal foundation for related researches.Methods The primary culture was undergone by the tissue digestion method.The VDSMCs were observed under the inverted microscope and identified with HE and anti α-SMA immunocytochemistry staining.The cell viability was also calculated by trypan blue dye exclusion.Continued with enzyme digestion and subculture,the growth curve of VDSMCs was drawn.Results The controlled experiment showed that Collagenase II was more efficient than Collagenase I with an optimal digestion time of 60 minutes.In addition,satisfying cell counts and viability were obtained by adequate pipetting and certain precipitation time,as well as proper culture environment.The morphology of VDSMCs which can be subcultured showed spindle shape under the microscopy and the cell growth showed the typical ＂peak and valley＂ characteristics with a ＂S＂ shaped growth curve.The cultured VDSMCs were positive to anti α-SMA immunocytochemistry staining with a purity of（92.6±4.3）%.Conclusion Tissue digestion method can serve as an ideal method for isolation and culture of rat vas deferens smooth muscle cells.