人源细胞系鉴定新技术的建立及应用

发布时间：2018-05-23 13:27

本文选题：细胞交叉污染 + 细胞身份鉴定　；参考：《武汉大学》2017年博士论文

【摘要】：细胞系作为生命科学、临床医学等研究的常用材料,其重要性不言而喻,但细胞的质量鉴定问题一直被许多研究者所忽视,细胞污染和细胞身份误判的现象非常普遍。近年来很多权威期刊和研究机构建议应该在研究论文出版发行或是申请基金之前进行相关细胞系的鉴定。虽然现在有很多新的方法能对细胞进行鉴定,但是基于短串联重复序列的DNA分型技术仍然是作为细胞鉴定的金标准。短串联重复序列(Short Tandom Repeat,STR)是由2-7个碱基对作为核心单位串联重复形成的一类DNA序列。由于具有广泛分布,信息量大,高度多态性并遵循孟德尔遗传规律,易于PCR扩增及分型等特征,STR技术已广泛用于亲子鉴定及人源细胞系交叉污染检测和身份鉴定。本论文利用21位点的STR分型新技术进行人源细胞系交叉污染检测和身份鉴定,共检测了来自28个研究单位的278株细胞系。检测结果发现,在278株细胞系中,46%的细胞存在交叉污染,Hep2和EJ细胞系还在错误身份下使用。73%(71株中52株)的交叉污染细胞来自于中国,占了交叉污染细胞总数的40%(129株中52株)。67%的污染源是HeLa细胞或者是HeLa细胞与其他细胞相互杂交的亚细胞株。肝癌细胞系HCCC-9810和退行性的肺癌细胞系Calu-6用9个位点的STR图谱数据(ATCC STR数据库)比对分析,显示了 0.89的相似性,而0.89的比对数据提示这两株细胞是同源细胞系。21位点的STR检测图谱分析和24个位点的SNP(Single Nucleotide Polymorphisms,SNP)检测结果均证明两株细胞是非同源细胞系,证明用21位点的STR新技术鉴定人源细胞系,数据更精确,结果更可靠。150株正确的细胞系展示了独特的分型图谱,排除了交叉污染,确认了身份。通过构建自主知识产权的细胞系STR数据库,为国内人源细胞系的STR分型提供比对标准,具有重要现实意义。
[Abstract]:The importance of cell line as a common material in life science and clinical medicine is self-evident, but the problem of cell quality identification has been ignored by many researchers. The phenomenon of cell contamination and misjudgment of cell identity is very common. In recent years, many leading journals and research institutions have suggested that cell lines should be identified before research papers are published or funds are applied. Although there are many new methods for cell identification, DNA typing based on short tandem repeats is still the golden standard for cell identification. Short Tandom repeat sequence (STR) is a kind of DNA sequence which is composed of 2-7 base pairs as core unit tandem repeats. Due to its wide distribution, large amount of information, high polymorphism and Mendelian genetic rule, PCR amplification and typing have been widely used in paternity testing, human cell line cross-contamination detection and identification. In this paper, a new 21 locus STR typing technique was used to detect the cross contamination and identification of human cell lines. A total of 278 cell lines from 28 research units were detected. The results showed that 46% of the 278 cell lines contained cross contamination of Hep2 and EJ cell lines. 52 or 67% of the total number of cross-polluted cells were HeLa cells or sub-cell lines of HeLa cells hybridizing with other cells. HCCC-9810 and Calu-6, a degenerative lung cancer cell line, were compared with each other by STR map data of 9 loci (ATCC STR database). The comparison data of 0.89 and 0.89 showed that the two cell lines were non-homologous cell lines. The results of STR analysis and 24 loci SNP(Single Nucleotide PolymorphismsSNPs analysis showed that the two cell lines were non-homologous. It is proved that the new technique of STR at 21 loci is more accurate in identifying human cell lines, and the results are more reliable. 150 strains of correct cell lines exhibit unique genotyping patterns, eliminate cross contamination and confirm their identities. It is of great practical significance to construct the STR database of human cell line, which can provide a comparison standard for STR typing of human cell lines in China.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R329.2

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