一株永生化口腔癌相关成纤维细胞系的建立及鉴定  

Establishment and identification of an immortalized oral cancer-related fibroblast line

作　　者：陈盛[1] 杨艳[1] 丁亮 叶传进 张磊[1] 夏舒 李慧玲 黄晓峰[1] Chen Sheng;Yang Yan;Ding Liang;Ye Chuanjin;Zhang Lei;Xia Shu;Li Huiling;Huang Xiaofeng(Department of Pathology,Affiliated Stomatological Hospital of Medical School of Nanjing University,Nanjing 210000,Jiangsu Province,China;Central Laboratory,Affiliated Stomatological Hospital of Medical School of Nanjing University,Nanjing 210000,Jiangsu Province,China)

机构地区：[1]南京大学医学院附属口腔医院病理科,江苏省南京市210000 [2]南京大学医学院附属口腔医院中心实验室,江苏省南京市210000

出　　处：《中国组织工程研究》2022年第24期3808-3813,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家青年科学基金项目(81902754),项目负责人:丁亮;江苏省自然科学基金项目(BK20190304),项目负责人:丁亮。

摘　　要：背景:肿瘤相关成纤维细胞系是构成肿瘤微环境的重要细胞成分,但是在原代培养过程中极易老化和表型丢失,进而显著降低肿瘤相关成纤维细胞功能相关研究的实验重复性。目的:旨在建立一种永生化的口腔癌相关成纤维细胞系,为探索口腔癌中肿瘤相关成纤维细胞功能提供稳定的细胞实验材料。方法:采用酶消化法分离、纯化人口腔癌组织来源肿瘤相关成纤维细胞,利用包装有人端粒酶反转录酶(pBABE-hygro-hTERT)的慢病毒感染原代肿瘤相关成纤维细胞,鉴定其形态及其标志物,然后进行细胞增殖、衰老功能检测,对原代和永生化肿瘤相关成纤维细胞进行染色体核型分析、细胞系身份鉴定。结果与结论:①成纤维细胞永生化后,其标志物α-SMA、PDGFRβ以及FSP-1表达没有发现显著改变;②即使在传代15代后,永生化成纤维细胞的增殖速率明显优于非永生化成纤维细胞;③β-半乳糖苷酶染色结果显示:随着细胞代数增加,与非永生化成纤维细胞相比,永生化成纤维细胞未见明显衰老;④永生化成纤维细胞染色体核型仍保留正常细胞的基本特征,而未发生恶性转化;⑤短串联重复序列鉴定结果显示,永生化成纤维细胞为原代细胞,与已知细胞数据库信息均无重合;⑥以上结果说明,成功建立了口腔癌来源的永生化成纤维细胞系,为口腔癌肿瘤微环境研究提供进一步的实验基础。BACKGROUND:Cancer-associated fibroblasts are important cell components that constitute the tumor microenvironment,but they are prone to aging and phenotype loss during the primary culture,which greatly reduces the experimental reproducibility of cancer-associated fibroblasts function-related research.OBJECTIVE:To establish an immortalized oral cancer-related cancer-associated fibroblast line and provide stable experimental cells for exploring the function of cancer-associated fibroblasts in oral cancer.METHODS:Enzymatic digestion method was used to isolate and purify cancer-associated fibroblasts from human oral cancer tissues,and the lentivirus packaged with human telomerase reverse transcriptase(hTERT)(pBABE-hygro-hTERT)was used to infect primary cancer-associated fibroblasts;and their morphology and markers were identified;cell proliferation and aging functions were tested.Furthermore,chromosome karyotype analysis and short tandem repeat analysis were performed on primary cancer-associated fibroblasts and immortalized cancer-associated fibroblasts for cell line identification.RESULTS AND CONCLUSION:(1)After fibroblasts were immortalized,the expression levels of their markersα-SMA,PDGFRβand FSP-1 were not significantly changed.(2)Even after 15 passages,their proliferation rate was significantly better than that of non-immortalized cells.(3)The results ofβ-galactosidase staining showed that with the increase of cell passage,compared with non-immortalized fibroblasts,immortalized fibroblasts did not present obvious senescence.(4)The karyotype of immortalized fibroblasts still retained the basic characteristics of normal cells without malignant transformation.(5)Short tandem repeat identification results demonstrated that immortalized fibroblasts were primary cells,and there was no overlap with the information in the known cell database.(6)These findings indicate that an immortalized fibroblast line derived from oral cancer has been successfully established,which provides a further experimental basis for studyi

关 键 词：肿瘤相关成纤维细胞 口腔鳞癌 原代细胞 永生化细胞系 细胞衰老 染色体核型鉴定 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]