rAAV／HCV核心抗原基因转染树突状细胞的免疫功能研究

目的 研究通过rAAV／HCV（hepatitis Cvirus，HCV）核心抗原基因（Coregene）转染树突状细胞（dendritic cell，DC）制备DC疫苗的免疫功能。方法分离外周血单个核细胞（DC前体细胞），以rAAV／Core／Neo病毒转染DC前体细胞（基因转染组），同时设293细胞裂解物刺激为对照组，转染12h后，均采用GM-CSF、IL-4、TNF-α诱导成熟。7天后收集细胞，流式细胞仪检测rAAV／Core／Neo病毒转染效率（即HCV核心抗原表达率）及DC表面标志CDl4、CD40、CD80、CD83、CD86的表达情况，混合淋巴细胞实验检测DC刺激自体T细胞增殖的能力，^51Cr释放法检测CTL对抗原阳性靶细胞的杀伤效率及特异性。结果rAAV／Core／Neo转染DC的效率超过90％，成熟DC高表达CD40、CD80、CD83、CD86，转染后的DC具有较强的刺激自体T细胞增殖能力，其CTL对HCV核心抗原阳性靶细胞具有很高的杀伤率及抗原特异性。结论rAAV／Core／Neo能够高效转染DC，转染后的DC能刺激自体T细胞增殖，使CTL对HCV核心抗原阳性靶细胞具有杀伤活性。

The immunological function study of DC transfected with rAAV/HCV core gene

Objective To study the immunological function of dendritic cell （DC） vaccine prepared by transfection with core gene of rAAV/hepatitis C virus. Methods Peripheral blood mononuclear cells （PBMCs） , a kind of DC precursors, were isolated. DC precursors were infected with rAAV/Core/Neo virus stock （gene transfeetion group）. On the other hand, in the control group, DC precursors were stimulated by the 293 cell lysate. Two groups were transfeeted for 12 hours, then followed an induction with GM-CSF, IL-4 and TNF-α. Seven days later, mature cells were collected and analyzed the transfective efficiency （HCV core antigen expression rate）. Furthermore, expressions of surface marker of DC including CD14, CD40, CD80, CD83, CD86 were determined with FACS, and ability to stimulation of T cell prolif- eration was measured by mix lymphocyte reaction. The rate of kill and specificity for CTLs against target cells with posi- tive antigen were measured with the 51Cr releasing analysis. Results The efficiency of rAAV/Core/Neo transfeetion DC was more than 90% , the transfection DC expressed high levels of CD40, CD80, CD83, CD86 and obtained the good biology activity in activating T cell. The CTLs showed high killing rate and antigen specificity to the HCV core antigen positive target cell. Conclusion DC can be transfected by rAAV/Core/Neo effectively. Thus, T cell proliferation can be stimulated with DCs after transfection. Furthermore, CTLs have the activity of kill against target cells with positive antigen.