人口腔癌相关成纤维细胞的分离培养及鉴定

The isolation,culture and identification of human oral carcinoma associated fibroblasts

目的:分离培养及鉴定口腔癌相关成纤维细胞(CAFs),为探究CAFs可能在肿瘤微环境促进口腔癌细胞的侵袭转移中发挥重要作用奠定基础。方法:用组织块培养法和酶消化法分离培养、纯化原代口腔癌相关成纤维细胞CAFs和口腔黏膜正常成纤维细胞(NFs);细胞免疫荧光技术及免疫蛋白印迹法对口腔CAFs和NFs进行鉴定。结果:口腔CAFs和NFs分离培养成功,口腔CAFs呈长梭形,达到一定密度时呈多层生长,排列紊乱;NFs呈多胞质突的扁平星状,无重叠生长现象,排列有一定方向性。上皮标志物细胞角蛋白18(CK18)免疫荧光染色在口腔CAFs和NFs均呈阴性,间质标志物波形蛋白(Vimentin)染色均呈阳性。α-平滑肌动蛋白(α-SMA)、成纤维细胞特异性蛋白-1(FSP-1),成纤维细胞激活蛋白(FAP)、血小板衍生生长因子受体α(PDGFR-α)在CAFs染色呈阳性,而在NFs中染色呈阴性。结蛋白(Desmin)在CAFs和NFs中染色均呈阳性。蛋白表达水平上,相对于NFs,CAFs中α-SMA、PDGFR-α蛋白表达呈升高趋势,FSP-1呈下降趋势,而FAP、Desmin在NFs和CAFs中表达无明显差异。结论:CAFs与NFs相比,在形态结构、生长方式、蛋白表达等方面均有明显差异。

Objective：To isolate and culture the oral cancer associated fibroblasts （ CAFs） and explore the possible role of CAFs in the invasion and metastasis of oral cancer cells in the tumor microenvironment. Methods：The tissue block culture method and enzymatic digestion method were used in the isolation, culture and purify of oral CAFs and normal mucous fibroblasts （ NFs） . And then the immunofluorescence assay and Western blot were used to identify the NFs and CAFs. Results：The oral CAFs were successfully isolated and cultured. They were long fusi form in shape, and the cells were in dense aggregations. The CAFs grew at certain point into multilayer structure and arranged in disorder. The cytokeratin 18 （ CK18） was negative in NFs and CAFs, while the Vimentin was positive. Theαsmooth muscle actin （αSMA） , fibroblast specific protein1 （ FSP1） , fibroblast activation protein （ FAP ） , and platelet derived growth factor receptor α（ PDGFRα） showed positive expression in CAFs compared to the NFs. The Desmin were expressed in NFs and CAFs. In the protein levels, the αSMA, PDGFRαwere significantly increased in CAFs as compared to that in NFs. In addition, the FSP1 was decreased in CAFs. However, there was no significant difference in the expression of Desmin and FAP in NFs and CAFs. Conclusions：In morphology, the oral CAFs and NFs were significantly different in growth and protein expression.