人B淋巴母细胞系体外培养方法的优化及其影响因素  

The Optimization of the Methods and the Influencing Factors of Establish Human B Lymphoblastoid Cell Lines

作　　者：刘敏娟[1] 孙静[1] 许云云[1] 臧志远[1] 刘柳[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,江苏苏州215007

出　　处：《苏州大学学报（医学版）》2010年第1期11-14,F0002,共5页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(30571690)

摘　　要：目的探讨体外建立健康人外周血B淋巴母细胞样细胞系(B-LCLs)的优化方法及其影响因素。方法用EB病毒感染健康人外周血中分离的单个核细胞(PBMC),加入CD40激发型抗体(5C11)、CpGDNA免疫调节基因序列以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性。光学显微镜下观察B-LCLs的形态特征,利用流式细胞术分析B-LCLs膜表面分子CD19的表达水平。结果PBMC经EBV感染3周后转化成永生化B-LCLs。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。CD40激发型抗体(5C11)及CpG免疫调节基因序列联合EBV感染人PBMC,明显地提高了转化效率,转化后的LCLs保持了成熟B淋巴细胞的生物学特征。结论CD40信号激发和CpG免疫调节基因序列联合运用能有效地促进EBV对人B淋巴细胞的转化及体外建系。Objective To explore optimized methods and the influencing factors of establishment of B lymphoblastoid cell lines（B-LCLs）derived from human PBMC in vitro.Methods The peripheral blood mononuclear cells（PBMCs）were isolated from healthy donors and incubated with EB virus（EBV）while agonist CD40mAb（5C11）,CpG DNA motifs for stimulating B lymphocytes and cyclospofin A（CyA）for inhibiting T lymphocytes were added at the same time.Morphological characteristics of immortalized B-lymphocytes were observed under optical microscope.Flow cytometry analysis was used to identify the expression of surface molecule CD19 on B-LCLs.Results PBMCs incubated with EBV for 3 weeks were transformed into immortalized B-LCLs.These immortalized cells accumulated into groups and were able to be cultured into the long term proliferation.The agonist CD40mAb and CpG DNA motifs markedly promoted the EBV transformation process.EBV-immortalized B-LCLs maintained the biological features of mature B lymphocytes.Conclusion The combination of CD40 signal activation and CpG DNA motifs can effectively promote the EB virus transformation into human B lymphocytes and cell line establishment.

关 键 词：EB病毒 CpG免疫调节基因序列 CD40信号 B淋巴母细胞系 

分 类 号：R392.2[医药卫生—免疫学] R331.144[医药卫生—基础医学]