喉癌相关成纤维细胞的分离培养与鉴定  

The Primary Culture and Identification of Laryngeal Carcinoma-associated Fibroblasts

作　　者：齐淑轶[1] 邓莅霏[1] 彭汉伟 王乐[1] QI Shuyi;DENG Lifei;PENG Hanwei(Jiangxi Cancer Hospital,Nanchang 330029)

机构地区：[1]江西省肿瘤医院,330029 [2]汕头大学附属肿瘤医院,515031

出　　处：《实用癌症杂志》2020年第6期875-878,共4页The Practical Journal of Cancer

基　　金：江西省自然科学基金项目(20132BAB205046)。

摘　　要：目的对喉癌相关成纤维细胞(CAFs)及正常喉成纤维细胞(NFs)进行分离、培养、纯化并鉴定、分析其生物学特性。方法采用组织块法和胶原酶消化法进行喉CAFs和NFs的原代培养。选择第3代喉CAFs及NFs,倒置显微镜下观察细胞的生长状态,HE染色光镜下观察细胞形态,免疫荧光法、qRT-PCR法及Western-blot法分别测定喉CAFs和NFs的CK、Vimentin、FAP及α-SMA的表达。CCK-8法检测喉CAFs和NFs的增殖活性,并绘制生长曲线。结果与喉NFs相比,喉CAFs细胞不规则,排列紊乱,且增殖活性明显增强;免疫荧光染色、qRT-PCR及western-blot检测发现,喉CAFs和NFs中CK均呈阴性表达,Vimentin、α-SMA及FAP则均呈阳性表达。结论通过组织块法和胶原酶消化法原代培养皆可获得高纯度的喉CAFs以供进一步研究之用。Objective To isolate and culture the laryngeal carcinoma associated fibroblasts(CAFs)and normal fibroblasts(NFs),and idenfy their biological characteristics.Methods The primary human laryngeal CAFs and NFs were obtained by tissue culture and digestion methods.The third generation cells were used for this study.The growth state of living cells was observed by inverted microscope.The morphological characteristics were identified by using HE staining.The expression of CK,Vimentin,FAP andα-SMA in the cells was examined by immunofluorescence staining,qRT-PCR and western-blot.Cell counting kit-8(CCK-8)assay was used to detect the proliferative activity and growth curves were drawn.Results As compared with NFs,the laryngeal CAFs were arranged in disorder,showed irregular shape and significantly enhanced proliferative activity.Determined by immunofluorescence staining,qRT-PCR and western-blot,both CAFs and NFs showed negative expression for CK,and positive expression for Vimentin,α-SMA and FAP.Conclusion The purified laryngeal CAFs are successfully obtained by both tissue culture and digestion methods for further research.

关 键 词：喉癌 癌相关成纤维细胞 原代培养 

分 类 号：R739.65[医药卫生—肿瘤]