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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 rat vas deferens smooth muscle cell isolation and culture method in vitro, which provides an ideal cell model for related research. Methods The isolated rat vas deferens smooth muscle cells were cultured in primary culture by tissue block digestion. The cells were identified by morphological observation, HE staining and anti-α-SMA immunocytochemical staining. Cell viability and total cell number were calculated by trypan blue staining, and the growth curve was drawn by trypsin digestion. Results The control experiment showed that digestive effect of collagenase Ⅱ was better than that of collagenase Ⅰ, and the optimal digestion time was 60 min. Meanwhile, the number of smooth muscle cells and the survival rate were obtained under the control of the number of blows, the precipitation time and the culture environment. Smooth muscle cells are typically spindle-shaped, elongated, passageable, and exhibit typical “peak-valley” features of smooth muscle cell culture with “S” shape. The anti-α-SMA immunofluorescence assay showed that the cultured cells were smooth muscle cells with a purity of (92.6 ± 4.3)%. Conclusion In vitro isolation and culture of rat vas deferens smooth muscle cells were successfully established by tissue digestion.