Establishment of Resting B Cell Model Infected with Latent EB Virus in Peripheral Blood
-
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.
-
Key words:
- Epstein-Barr virus /
- Latent infection /
- CD5+B /
- Resting B cell
-
-
[1] Price AM,Tourigny JP,Forte E,et al. Analysis of Epstein-Barr virusregulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1- mediated NF-κB activation[J].J Virol,2012,86(20):11096-106. [2] Rasul AE, Nagy N, Sohlberg E, et al. Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells[J]. J Immunol Methods, 20 12,385(1-2):60-70. [3] Leonard S, Wei W, Anderton J, et al. Epigenetic and transcriptional changes which follow Epstein-Barr virus infection of germinal center B cells and their relevance to the pathogenesis of Hodgkin’s lymphoma[J]. J Virol, 2011,85(18):9568-77. [4] Kalla M, Hammerschmidt W. Human B cells on their route to latent infection--early but transient expression of lytic genes of Epstein-Barr virus[J]. Eur J Cell Biol, 2012 ,91(1):65-9. [5] Jin ZS,An JD,Song HL,et al. EB virus latent infection of mantle cell lymphoma model[J]. Zhongguo Zhong Liu Lin Chuang,2012,39 (1):1-4. [金在顺,安锦丹,宋华林,等. EB 病毒潜 伏感染的套细胞淋巴瘤模型研究[J]. 中国肿瘤临床, 2012,39 (1) :1-4.] [6] Gregorovic G, Bosshard R, Karstegl CE, et al. Cellular gene expression that correlates with EBER expression in Epstein- Barr Virus-infected lymphoblastoid cell lines[J]. J Virol, 20 11,85(7):3535-45. [7] Hayashi K, Jin Z, Onoda S, et al. Rabbit model for human EBV-associated hemophagocytic syndrome (HPS): sequential autopsy analysis and characterization of IL-2 dependent cell lines established from herpesvirus papio-induced fatal rabbit lymphoproliferative diseases with HPS[J]. Am J Pathol, 2003,162 (5) :1721-36. [8] Iwakiri D, Takada K. Role of EBERs in the pathogenesis of EBV infection [J]. Adv Cancer Res, 2010,107:119-36. [9] Halder S, Murakami M, Verma SC, et al. Early events associated with infection of Epstein-Barr virus infection of primary B-cells[J]. PLoS One,2009, 4(9):e7214.
计量
- 文章访问数: 1827
- HTML全文浏览量: 457
- PDF下载量: 1284
下载:
