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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 ,这将有助于开展以DC为基础的慢性乙肝的免疫治疗
Objective: To investigate retrovirus-mediated transfer efficiency of hepatitis B virus core antigen (HBcAg) gene in bone marrow-derived dendritic cells (DCs) and its effect on DC maturation and function. Methods: The myeloid progenitor cells were infected with the recombinant retroviral vector containing HBVC gene. The infected bone marrow cells were cultured in the presence of GM CSF and IL 4 to obtain mature DCs. PCR, RT PCR analysis of the integration and transcription of the target gene; analysis of HBcAg expression and gene transfer efficiency by Western blot and flow cytometry, as well as detection of DCCD80 and MHC class Ⅱ molecules before and after infection and secretion of IL 12 capacity changes; by gene After the transfer of DC and lymphocytes were mixed culture to detect its ability to induce CTL in vitro. Results: Retroviral infection did not affect the maturation of bone marrow-derived DCs and had no effect on the expression of surface molecules CD80 and MHC class II molecules and the ability to secrete IL-12. The target gene can be integrated into the genomic DNA of DC and can be transcribed and translated. About 28% of DCs express HBcAg, and infected DCs induce T cell responses in vitro. CONCLUSIONS: Retroviruses can efficiently transfer HBVC genes into bone marrow-derived DCs with no significant effect on the maturation and function of DCs and induce CTL responses, which will contribute to the development of DC-based immunizations against chronic hepatitis B treatment