建立不同民族永生细胞株质量控制方法的探讨

目的以建立普米族、独龙族和怒族永生细胞为基础,探讨EB病毒转化细胞、细胞培养、冻存、复苏以及支原体检测等建立永生细胞株的技术要点及质量检测方法.方法采用EB病毒转化技术建立3个民族永生细胞株;培养法和PCR法对细胞株进行支原体污染检测;染色体G显带及核型分析细胞株的遗传稳定性.结果成功建立了3个民族B淋巴细胞永生细胞株,转化率分别为98%、86%和76%.对已建株保存的3个民族的永生细胞进行复苏培养,复苏成活率为100%.支原体污染检测均为阴性.染色体计数和G带分析显示,细胞株经早期传代培养后,仍然保持二倍体特征,未发现染色体结构畸变.结论本研究中传代培养、细胞冻存、细胞复苏和支原体污染防范等一整套技术过程是满足建库要求的.同时为大规模永生细胞库的建立和进行相应的质量监测提供了科学的依据.另外,本研究还对一些影响转化的因素和可能的机理进行了探讨.

Study on quality control of immortal lymphoblastoid cell lines derived from ethnic groups

Objective: To systematically study the quality control of a resource panel of Epstein-Barr virus(EBV) transformed immortal lymphoblastoid cell lines (LCLs) from Pumi, Dulong and Nu minorities by subculturing, cryogenic preservation, culture recovery and mycoplasmal contamination prevention. Methods: Establish an immortal cell panel by EBV transformation of lymphocytes from the three minoritie, detect mycoplasmal contamination by culture and PCR, and determine cell genetic stability by chromosomal G bands and karyotype analysis.Results: A resource panel of lymphoblastoid cell line from the three minorities , was successfully established with a transformation rate of 98%,86% and 76% respectively. Culture recovery rate was 100%. The LCLs were sampled to detect mycoplasmal contamination by polymerase chain reaction-based method and culture assay, showing a result of negative infestation. Furthermore, the microscopic evidences of cell growth patterns and cellular morphology, and chromosomal structures confirmed a cellular and genetic stability. Conclusion: The processes pertaining to subculturing, cryogenic preservation, culture recovery and mycoplasmal contamination prevention were satisfactorily adapted in establishment of the cell line panel. The LCLs could provide a samples resource for studies related to genomic diversity and history of modern human populations and offer the scientific basis for a larger panel and its quality determination. In addition, the possible mechanisms and some factors involved in cell transformation were discussed in the research.