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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 rat primary culture model of rat meningeal mesothelial cells (RMMC). The primary cells were harvested by mechanical pipetting and 0.25% trypsin-0.01% EDTA digestion, with multiple differential adhesion and passage. After three passages, the general structure of cells was observed by inverted phase contrast microscope. The ultrastructure of the cells was observed by scanning electron microscopy (SEM). The morphology of cells was observed by hematoxylin and eosin (HE) staining. Cell immunofluorescence and immunocytochemistry were used to identify the cells. The results showed that the primary cells isolated and cultured were polygonal under the inverted microscope and arranged in a paving way when confluent. Large numbers of microvilli were observed on the surface of the cells under SEM. Immunofluorescence staining showed that the cultured cells were positive for cytokeratin antigen. Cytochemical results showed that the cultured cells were positive for cytokeratin and vimentin antigen, negative for factor Ⅷ related antigen, and the purity of cultured cells was over 96%. Conclusion: The successful establishment of the RMMC culture model in this experiment can provide an ideal cell model for the study of the formation mechanism of meningeal fibrosis.