改良法体外培养大鼠脑膜间皮细胞

Improvement on culture in vitro of rat menigeal mesothelial cell

建立大鼠脑膜间皮细胞(rat meningeal mesothelial cells,RMMC)的体外原代培养模型。用机械吹打法和0.25%胰酶-0.01%EDTA轻消化法收集原代细胞,多次差速黏附处理及传代。传三代,分别进行倒置相差显微镜观察细胞大体结构、扫描电镜(SEM)观察细胞超微结构、苏木素伊红(HE)染色法观察细胞形态、细胞免疫荧光法及免疫细胞化学法鉴定细胞。结果显示:分离培养的原代细胞在倒置显微镜下为多边形,汇合时呈铺路石样排列;SEM下细胞表面可见大量密集的微绒毛;免疫荧光染色结果显示培养细胞呈细胞角蛋白抗原阳性;免疫细胞化学结果显示培养细胞呈细胞角蛋白和波形蛋白抗原阳性,第Ⅷ因子相关抗原阴性,培养细胞的纯度达96%以上。结论:本实验成功建立了RMMC培养模型,可为脑膜纤维化的形成机制研究提供理想的细胞模型。

To establish a reproducible model for culture of rat meningeal mesothelial cells （ RMMCs）. Primary cells were collected by mechanical blow and gently enzymatic-EDTA disaggregated methods, and then repeat adhering treatment by different speed and passage. After three passages, morphological changes were detected through downward microscope during cell culture. The ultrastructure was observed by scanning electron microscopy （SEM）. Morphologic types were observed by HE staining. Isolated cells were characterized by immunofluorescence and immunohistochemical analysis. The results showed that cultured cells were multipolar and presented a cobblestone-like when they reached degeneration confluence,and abundant microvilli on the surface of the cells were observed under SEM. Immunofluorescence studies showed that the cultured ceils were positive staining for cytokeratin. Immunohistochemical studies showed that the cells were positive staining for cytokeratin and vimentin,but negative staining for factor Ⅷ associated antigen. The purity of cells were over 96%. The above results suggest that the model of RMMCs has been successfully established and it will provide the ideal cell model for the research on the mechanism of meninges fibrosis.