关键词: 免疫组织化学, 结肠, 成纤维细胞, HE染色, 原代培养, Wistar大鼠

Abstract: 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(S100A4) and E-cadherin in the cells.The fibroblasts were identified by comparison with the smooth muscle A7R5 cells of thoracic aorta of the rats. Results:The fibroblasts began to climb from the tissue at the 8th day,they began to proliferate at the 12th 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 of α-SMA,S100A4 and E-cadherin were negative.The expression of S100A4 in the smooth muscle A7R5 cells of thoracic aorta of the rats in control group was negative,and the expressions of α-SMA,Vimentin and E-cadherin were positive. Conclusion:The rat colon fibroblasts can be successfully cultured by trypsin combined with explant culture method.

Key words: colon, primary culture, HE staining, fibroblasts, Wistar rats, immunohistochemistry