Epstein-Barr病毒转化免疫性血小板减少症患者外周血B淋巴细胞并建立永生化细胞株  

Establishment of immortalized B lymphoblastoid cell lines from the patients with immune thrombocytopenia by using Epstein-Barr virus

作　　者：贺怡子 孙楠楠[1] 刘素芳[1] 张广森[1] 

机构地区：[1]中南大学湘雅二医院血液内科,长沙410011

出　　处：《临床血液学杂志》2016年第1期37-40,共4页Journal of Clinical Hematology

基　　金：国家自然科学基金(No:81470323)

摘　　要：目的:体外建立正常人及免疫性血小板减少症(ITP)患者永生化的B淋巴母细胞系(iBLCLs)并鉴定其生物学活性,为研究ITP的发病机制奠定基础。方法:采用B958细胞产生的EB病毒(EBV)感染正常人及ITP患者外周血单个核细胞(PBMCs),获得永生化细胞株后,光学显微镜观察iBLCLs细胞形态学特征,RT-PCR技术检测EBV中潜伏膜蛋白1(LMP-1)的表达,流式细胞术分析iBLCLs细胞周期及检测细胞膜表面CD19分子标记的表达。结果:5例正常人PBMCs均成功转化为iBLCLs;5例ITP患者中3例PBMCs转化成iBLCLs,其余2例PBMCs在转化16天左右开始退化未能成功建株。ITP患者成功转化所需时间明显长于正常人。所有iBLCLs均表达LMP-1基因序列和细胞膜表面CD19分子。结论:PBMCs感染EBV后建立的iBLCLs保持了成熟B淋巴细胞的生物学活性特征,在转化过程中第16天左右存在一个"危机期",未感染EBV的细胞,细胞数量进行性减少并死亡。部分ITP患者的PBMCs因不能度过这个"危机期"而转化失败。如何改进实验条件以提高ITP患者PBMCs中B淋巴细胞建株的成功率有待进一步研究,从而为体外研究ITP免疫异常机制提供靶细胞。Objective：To establish immortalized B lymphoblastoid cell lines（iBLCLs）from peripheral blood mononuclear cells（PBMCs）of healthy human and immune thrombocytopenia（ITP）patients in vitro and study its biological function.Method：PBMCs were isolated from ITP patients and normal controls,and then incubated with Epstein-Barr virus（EBV）.Then optical microscope was used to observe iBLCLs.The gene expression of EBV encoded latent membrane protein 1（LMP-1）in iBLCLs were detected by RT-PCR.The cell cycle and expression of surface molecules CD19 in iBLCLs were analyzed by flow cytometry.Result：We successfully got eight strains of iBLCLs,5from heathy controls and 3from ITP patients,2strains from ITP patients failed to get establishment due to degradation since the 16 th day.LMP-1and CD19 expression were positively detected in all iBLCLs.Conclusion：EBV-immortalized iBLCLs can maintain the biological features of mature B lymphocytes.There is a crisis period in the process of transformation,some of the cells from ITP patients could not survive.The experimental conditions in which we can offer assistance to get through the crisis needs to be further studied.

关 键 词：免疫性血小板减少症 EB病毒 永生化细胞株 

分 类 号：R558.2[医药卫生—血液循环系统疾病]