改良组织块消化法建立大鼠气道平滑肌细胞模型

An improved model of cultured rat airway smooth muscle cells By mixed tissue-piece digestion method

目的：建立改进大鼠气道平滑肌细胞（ASMC）的体外培养方法，为相关研究提供实验材料。方法：将组织块连续贴壁进行细胞原代培养，胰酶消化传代培养，差速贴壁进行细胞纯化，形态学及免疫细胞化学染色法进行细胞鉴定。MTT法检测PDGF-BB诱导的ASMC增殖。结果：成功培养大鼠ASMC，以改良组织块消化法最为理想。第四代平滑肌细胞纯度可达95％以上。相差显微镜下培养细胞呈典型“峰谷状”生长。免疫荧光化学染色显示特异性平滑肌肌动蛋白阳性表达。随着PDGF浓度的升高（2-80ng／ml），MTT比色A490值呈上升趋势。与对照组相比较，80ng／ml、20ng／ml PDGF—BB组有统计学意义（P〈0．01）。结论：改良组织块消化法可缩短培养周期，在充分利用标本的基础上获得大量气道平滑肌细胞。

Objective： To establish an improved cultured model of rat airway smooth muscle cells （ASMC） in vitro. Methods： The primary and transfer cells were cultured by mixed tissue-piece digestion inoculation. The cells were purified by natural passage transfer and differential adhesion. The cells were identified by morphological characteristics and immunocytochemistry. Results： The purity of the fourth passage ASMCs was over 95%. ASMC possessed ＂peak and valley＂ characteristics in phase contrast microscope. Immunocytochemical staining with specific mAb against rat SMAα-actin demonstrated these cells as positive. A490 value of ASMC by MTT assay increased with the elevation of PDGF-BB concentration（2-80 ng/ml）. Compared with control group, A490 value of 20ng/ml PDGF group and 80ng/ml PDGF group were increased significantly （P 〈0.01）. Conclusion： It is a simple and reliable model for obtaining highly purified airway smooth cells by improved tissue-piece digestion inoculation.