逆转录病毒介导乙型肝炎病毒C基因在树突状细胞中的转移  

Retroviral-mediated transfection of hepatitis B virus core gene into bone marrow-derived dendritic cells

作　　者：丁传林[1] 姚堃[1] 张天泰[2] 彭光勇[1] 

机构地区：[1]南京医科大学微生物学与免疫学系,江苏南京210029 [2]兰州医学院第一附属医院,甘肃兰州730000

出　　处：《细胞与分子免疫学杂志》2003年第3期299-301,304,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省重点实验室开放基金资助 (No.k2 0 30 )

摘　　要：目的 :探讨逆转录病毒介导乙型肝炎病毒核心抗原 (HBcAg)基因在骨髓来源的树突状细胞 (DC)的转移效率 ,以及对DC成熟和功能的影响。方法 :用含有HBVC基因的重组逆转录病毒载体 ,感染处于分裂期的小鼠骨髓祖细胞 ,感染后的骨髓细胞在GM CSF和IL 4存在的条件下继续培养获得成熟的DC ,用PCR、RT PCR ,分析目的基因的整合与转录 ;用Westernblot和流式细胞仪 ,分析HBcAg的表达及基因转移效率 ,以及检测感染前后DCCD80和MHC Ⅱ类分子的表达以及分泌IL 12能力的变化 ;通过将基因转移后的DC与淋巴细胞进行混合培养 ,检测其体外诱导CTL的能力。结果 :逆转录病毒感染后并不影响骨髓来源的DC成熟 ,对其表面分子CD80和MHC Ⅱ类分子的表达和分泌IL 12的能力没有影响。目的基因能整合到DC的基因组DNA中 ,并能转录和翻译。约 2 8%的DC能表达HBcAg ,感染后的DC体外能诱导T细胞应答。结论 :逆转录病毒能有效地将HBVC基因转移到骨髓来源的DC中 ,对DC的成熟和功能无明显影响 ,并能诱导CTL应答 。AIM: To evaluate the transfection efficiency of recombinant retrovirus vector bearing hepatitis B virus(HBV) core gene to bone marrow derived dendritic cells(DCs) and the capability of these DCs to induce cytotoxic T lymphocyte(CTL) response. METHODS: C57BL/6 mice bone marrow cells were stimulated with recombinant mouse granulocyte macrophage colony stimulating factor(rmGM CSF) and interleukin 4(rmIL 4) for 6 days. Expanding DC progenitors were transfected by retrovirus vector containing HBV core gene. Integration and transcription of HBV core gene were determined by PCR and RT PCR, respectively. Expression of HBcAg was analyzed by fluorescence activated cell sorter (FACS) and Western blot. Cytokines were quantified by enzyme immunoassay. The expressions of CD80 and MHC class Ⅱ molecules on DCs were measured by FACS. Generation of CTLs in mixed leukocyte reaction were determined by LDH release assays. RESULTS: Transfected bone marrow cells were capable of differentiating into DCs in vitro at the presence of rmGM CSF and rmIL 4. The result of PCR and RT PCR showed that the HBV core gene was integrated into the genome of infected DCs. Western blot analysis showed that HBV core gene was expressed in DCs. Transfection rate was 28% determined by FACS. Retroviral transfection had no influence on expressions of CD80 and MHC class Ⅱ molecules, as well as IL 12 production. HBcAg specific CTLs could be generated by using transfected DCs as antigen presenting cell (APC). CONCLUSION: Retroviral transfected myeloid DC progenitors could efficiently express HBcAg, without significant change on development and function of DCs, which lays a solid foundation for immunotherapy of chronic hepatitis B.

关 键 词：逆转录病毒 乙型肝炎病毒 树突状细胞 基因转移 

分 类 号：Q754[生物学—分子生物学] R373.2[医药卫生—病原生物学]