EB病毒转化人外周血B淋巴细胞的影响因素研究

目的探讨人类疱疹病毒(EBV)转化外周血B淋巴细胞试验的影响因素。方法用B95-8细胞制备EBV,实时荧光定量-PCR(realtime-PCR)法测定EBV滴度以确保病毒有效性,Ficoll密度梯度离心法分离外周血单个核细胞,研究环孢霉素A(CSA)的浓度、CSA加入时间、淋巴细胞浓度3个因素对EBV转化外周血B淋巴细胞试验的影响,选择最优化条件对三氯乙烯接触工人的B淋巴细胞进行转化。结果制备的病毒滴度分别为5.45×106/ml和1.06×106/ml。不同浓度CSA(0.2、1和2μg/ml)作用以及不同加入时间(同时加入或2h后)B淋巴细胞均成功永生化;正常人B淋巴细胞转化时淋巴细胞浓度不低于2×106/ml时方能成功永生化,而接触三氯乙烯工人的淋巴细胞在1×106/ml浓度下亦转化成功。不同条件下转化成功的B淋巴细胞永生化细胞系在形态上无明显差别,显微镜下可见B淋巴细胞体积增大并聚集成团。结论在建立B淋巴细胞永生化细胞系的过程中,CSA浓度和加入时间对转化结果未见影响,而淋巴细胞与EBV接触时的细胞浓度是影响实验成败的关键因素,接触三氯乙烯工人的B淋巴细胞在1×106/ml浓度下亦转化成功,推测与淋巴细胞致敏状态有关。

Study on the influence factors of human peripheral blood B lymphocyte transformation by epstein-barr virus

Objective To investigate the influence factors of human peripheral blood B lymphocyte transformation by epstein-barr virus(EBV). Methods EB virus was obtained from B95-8 supernatant and quantitated by quantitative PCR amplification. The concentration of cyclosporine A(CSA) concentration of lymphocytes and different time of adding CSA to culture media were selected to examine the effects on the lymphocyte transformation, and the condition of lymphocyte transformation of worker exposed to triehloroethylene was further confirmed. Results The titer of two virus samples were 5.45 ×10~6 and 1.06 ×10~6 copies/ml. Peripheral blood mononuclear cells (PBMCs) was transformed by using CSA at different concentration and adding time to culture fluid. The concentration of PBMCs being transformed was 2×10~6/ml for normal people, but 1 ×10~6/ml for worker exposed to triehloroethylene. Cell transformation was confirmed by the gradual increase of cell size and cellular clusters. Conclusion The results suggest the key issue is PBMCs being transformed at a concentration of 2 ×10~6/ml, rather than using CSA at different concentration and adding time to culture fluid. The PBMCs at concentration of 1 ×10~6/ml for worker exposed to trichloroethylene were transformed, which may be associated with sensitized status of lymphoeytes.