大鼠结肠原代成纤维细胞的分离、培养和鉴定

Isolation,culture and identification of primary fibroblasts from rat colon

目的:建立简单高效的大鼠结肠成纤维细胞体外原代培养和鉴定方法,为进一步研究结肠纤维化疾病提供细胞模型。方法:取成年雄性Wistar大鼠结肠组织,采用组织块贴壁法进行成纤维细胞体外原代培养,胰酶联合差速组织块贴壁培养法分离成纤维细胞,HE染色观察细胞形态表现,免疫组织化学法观察细胞中波形蛋白(Vimentin)、α平滑肌肌动蛋白(α-SMA)、S100钙结合蛋白A4(S100A4)和E钙黏蛋白(E-cadherin)的表达,并与大鼠胸主动脉平滑肌A7R5细胞对比进行鉴定。结果:结肠组织块贴壁培养8d后成纤维细胞爬出,12d进入增殖时期,15~20d基本长满。倒置显微镜下观察,细胞呈扁平多突的纺锤形或星形;HE染色,成纤维细胞核大,卵圆形,着色浅,核仁明显。免疫组织化学染色,成纤维细胞Vimentin呈阳性表达,α-SMA、S100A4和E-cadherin均呈阴性表达;对照组大鼠胸主动脉平滑肌A7R5细胞S100A4呈阴性表达,α-SMA、Vimentin和E-cadherin均呈阳性表达。结论:采用胰酶联合差速组织块贴壁培养法成功原代培养出大鼠结肠成纤维细胞。

Objective：To establish a simple and efficient method for primary culture and identification of the rat colon fibroblasts in vitro,and to provide cell model for the further study on colon fibrotic diseases.Methods：The colon tissue of adult male Wistar rats was selected.The rat colon fibroblasts were cultured by tissue explant method,and isolated by trypsinzation combined with different speed attaching.The morphology of fibroblasts was observed by HE staining.Immunohistochemistry staining was used to observe the expressions of Vimentin,α-smooth muscle actin（α-SMA）,S100 calcium-binding protein A4（S100 A4）and E-cadherin in the cells.The fibroblasts were identified by comparison with the smooth muscle A7 R5 cells of thoracic aorta of the rats.Results：The fibroblasts began to climb from the tissue at the 8 th day,they began to proliferate at the 12 th day of the culture,and grew throughout the culture flasks at the days 15-20.The fibroblasts were flat polygonal spindle or starlike under inverted microscope.The HE staining results showed that the nuclei of fibroblasts were large,oval and pale colored and had prominent nucleoli.The results of immunohistochemistry showed that the expression of Vimentin in fibroblasts was positive,and the expressions ofα-SMA,S100 A4 and E-cadherin were negative.The expression of S100 A4 in the smooth muscle A7 R5 cells of thoracic aorta of the rats in control group was negative,and the expressions ofα-SMA,Vimentin and E-cadherin were positive.Conclusion：The rat colon fibroblasts can be successfully cultured by trypsin combined with explant culture method.