人膀胱癌组织中成纤维细胞的培养和鉴定

The culture and identity of the fibroblast in the human bladder carcinoma tissue

目的:探索人膀胱癌组织中成纤维细胞的培养和鉴定方法,为其功能的研究奠定基础。方法:分别应用组织块法、消化法进行人膀胱癌组织中成纤维细胞的原代培养,细胞铺满培养瓶底后首次传代,通过胰酶消化法和反复贴壁法进行成纤维细胞的纯化;通过倒置显微镜观察细胞形态、透射电镜观察细胞内部结构和免疫组化方法检测细胞膜波形蛋白(vimentin)、角蛋白(keratin)表达情况对细胞进行鉴定。结果:组织块法原代培养约1周可见纤维样细胞从组织块周围游出,消化法4天后可见细胞贴壁,均于5周左右细胞基本长满培养瓶底。首次传代时应用胰酶消化法进行成纤维细胞的初次纯化,第二次转种时先后应用酶消化法和反复贴壁法再次进行细胞纯化,第3、4代基本可获纯的含单个核的长梭形纤维样细胞。取第5、6代状态良好的细胞常规准备后进行透射电镜检查和免疫组化染色:电镜提示细胞内可见明显的粗面内质网、高尔基体和(或)微丝结构,线粒体数量少,分布不均;免疫组化结果为vimentin染色阳性,keratin染色阴性。结论:只要给细胞创造良好的生长条件并正规操作,组织块法和消化法均可成功地进行人膀胱癌组织中成纤维细胞的原代培养;因与恶性肿瘤细胞相比,成纤维细胞的优势生长并不明显,故细胞的纯化工作极为重要。

Objective：To investigate the method of the culture and identity of the fibroblast in the human blad der carcinoma tissue. Methods：We had cultured in vitro the fibroblast in the human bladder carcinoma tissue with digestion method or tissue mass method, had used digestion with trypsin and adherence repeatedly method to depurate the fibroblast after the cell overgrew the bottom of the culture flask. The identity of the fibroblast was through the obserwltion of the shape of the cell with inverted microscope and the cellular organ with electromicroscope, the expression of vimentin and keratin in the cellular membrane with immunocytochemistry. Results： The fiber like cell emigrated from the tissue mass one week after the primary culture in tissue mass method, the adher ence of the cell could be watched 4 days after digestion method primary culture. The cell＇s overgrowth of the bottom of culture flask needed about 5 weeks. The first time depuration of the fibroblast was performed in the first generation cells with digestion methods, and the second time depuration was through the digestion method and adherence repeatedly by turns in the second generation cells, and we had gotten simple fusiform shape fiber like cell after these operation in the third or forth generation cells. The observation of the cellular organ and immunocytochemitry were performed in the fifth of sixth generation cells, We could watch many rough endoplasmic reticulum, golgiosome, microfilament structure and little bioblast in these fiber like cells, positive staining of vimentin and negative staining of keratin in the cellular membrane. Conclusions：The primary culture of the fibroblast in the hu man bladder carcinoma tissue can be performed successfully with digestion method or tissue mass method when we give the cell suitable grow condition and operate normally. The depuration of the cell is very important for the dominant growth of the fibroblast is not remarkable when compared with carcinoma cell.