末梢血中EB病毒潜伏感染的静息B细胞模型的建立
Establishment of Resting B Cell Model Infected with Latent EB Virus in Peripheral Blood
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摘要: 目的 应用EB病毒(EBV)感染末梢血CD5+B细胞，建立体内储存EB病毒的静息B细胞模型。方法 应用磁珠技术分离脐胎血中的CD5+B细胞；从新酶素耐性的Atada-EBV细胞中提取EB病毒对CD5+B细胞进行转染制备CD5+B/EBV细胞；流式细胞仪、Western blot法检测CD5+B/EBV细胞的病毒免疫表型和基因表型；免疫荧光双重染色BrdU、Ki67和EBNA2检测CD5+B/EBV细胞增殖活性。结果 流式细胞仪检测显示CD5+B/EBV细胞病毒免疫表型CD20、CD80、CD38和CD86表达增强，CD23 没有表达；CD5+B/EBV细胞EBNA2和LMP1病毒相关潜伏感染基因显著增高；免疫荧光染色检测EBNA2阳性的CD5+B/EBV细胞可见BrdU阴性细胞。经因子激活CD5+B/EBV后可见Brdu阴性细胞中有Ki67阳性细胞存在，而在EBNA2阳性细胞中可见Ki67阴性细胞，验证CD5+B/EBV细胞中静息B细胞的存在。结论 本研究建立的CD5+B/EBV细胞具有体内EBV潜伏感染的静息B细胞的特征。Abstract: Objective To establish the ideal model of resting B cell containing Epstein-Barr virus in vitro by EBV latent infected CD5+ B cells of peripheral blood. Methods CD5+B cells were obtained by mouse IgG magnetic beads. We took advantage of neomycin resistance to reorganize EBV genes (NeoR EBV) on the CD5+B cells to transfect G418 as the selection antibiotics, and labeled by BrdU. The immune phenotypes, gene phenotypes and proliferation of CD5+B/EBV cells were detected by Western blot, double immunofl uorescence and fl ow cytometry, respectively. Results In CD5+B/EBV cells, expression of EBNA2 and LMP1 were signifi cantly increased. Also, the expression levels of immune phenotype of CD20, CD80, CD38 and CD86 were signifi cantly increased, however, CD23 was not changed much in CD5+B/EBV. BrdUnegative cells were visible in EBNA2-positive cells. After cell factor activated, Ki67-positive cells were visible in Brdu-negative cells. However, Ki67-negative cells were visible in EBNA2-positive cells, resting B cells were exist in CD5+B/EBV cells. Conclusion CD5+B/EBV cell established in this study were characterized with resting B cells latently infected by EBV.
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