国内建立的人肿瘤细胞系的身份认证问题及对策

doi:10.16098/j.issn.0529-1356.2019.02.010

摘要/Abstract

摘要：

目的为国内建立的人肿瘤细胞系进行身份认证，确认这些细胞系使用的可靠性。方法收集、整理国内实验室建立的人肿瘤来源细胞系，提取细胞的基因组DNA，PCR扩增其中的多个短串联重复序列（STRs）位点后进行毛细管电泳，分析获得细胞的STR图谱作为细胞系的DNA指纹，然后与原始组织或国际数据库中已有细胞的STR谱进行比对，通过匹配度的高低判断该细胞是否被其他细胞交叉污染。对怀疑HeLa细胞污染的肿瘤细胞，检测其基因组中是否有人乳头瘤病毒18型（HPV-18）病毒插入片段。对怀疑T细胞白血病细胞污染的，则检测T细胞的各种特性（表面标志），根据表面标志表达情况判断是否为错误细胞。结果本研究共收集了37种国内建立的肿瘤细胞系46个样品。本中心建立的6株细胞系均STR正确。其他单位建立的细胞系中有8株系正确，有23（62.2%）株系认定为错误细胞，人宫颈癌细胞HeLa是主要污染源；另外发现有些国内自建的细胞系被人结直肠癌细胞HCT-8、宫颈鳞癌细胞SiHa、T细胞白血病细胞CCRF-CEM及大鼠细胞污染。结论国内建立的肿瘤细胞很高比例地发生了细胞间的交叉污染，科研人员在实验前一定要首先进行细胞身份认定，或者从国家实验细胞资源共享服务平台选择细胞。另外，建立细胞系时保留原始组织用于后续细胞系的身份认证。

关键词: 短串联重复序列分型, 交叉污染, 特性鉴定, 聚合酶链反应, 肿瘤细胞, 细胞培养, 人

Abstract:

Objective To authenticate the identity of human tumor cell lines established in China and make sure the authenticated cell lines will be used in science and technology research in China. Methods We collected and extracted genomic DNA of those tumor cells, amplify short tandem repeat (STR) loci by PCR and performed electrophoretic analysis to get STR profiles (as the DNA finger print of the cell line). Comparing their STR profiles against international STR database and those of their donor tissues, crosscontaminated cell lines will be excluded from the service list. PCR detection of human papilloma virus type 18（HPV-18）fragment were performed to double check HeLa crosscontaminated cell lines. Suspected cells contaminated by T leukemia cells were checked by detection characteristics of T cells, such as expression of some surface markers. Results Altogether 46 samples of 37 human tumor cell lines were collected from different labs. All 6 cell lines established in our center showed identical STR profile with their original tissue or primary culture. Among the tumor cell lines established by others 8 cell lines showed unique STR profile, indicating authentic. The other 23 (62.2%) of them showed identical STR with HeLa or with high evaluation value (EV) value, HPV fragment were found in their genomic DNA indicating replacement by or hybrid with HeLa. Some tumor cell lines established in China were cross-contaminated by human colorectal cancer cell line HCT-8, cervical squamous cancer line SiHa, T leukemia cell line CCRF-CEM and a rat cell line. Conclusion A large proportion of human tumor cell lines established by Chinese scholars were cross-contaminated. So scientists must first do authenticate these cell lines before experiments or obtain cell lines from China Infra-structure of Cell Line Resource. Furthermore, when trying to establish cell lines, preserve original tissue/DNA for future authentication.

Key words: Short tandem repeat DNA typing, Cross-contamination, Characterization, PCR, Tumor cell, Cell culture, Human

卞晓翠刘玉琴杨振丽冯海凉. 国内建立的人肿瘤细胞系的身份认证问题及对策[J]. 解剖学报, 2019, 50(2): 201-210.

BIAN Xiao-cui LIU Yu-qin YANG Zhen-li FENG Hai-liang. Troubles and answers of identity authentication of human tumor cell lines established in China[J]. Acta Anatomica Sinica, 2019, 50(2): 201-210.

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