目的 构建丙型肝炎病毒(HCV)多细胞毒性T淋巴细胞(CTL)表位树突状细胞(DC)疫苗,观察其体外刺激的T细胞反应,为下一步做体内免疫实验提供一定的资料.方法 构建和制备出含绿色荧光蛋白(GFP)标签的HCV两个CTL表位的重组腺病毒,感染DC,直接在荧光显微镜下或用流式细胞仪检测其感染率;RT-PCR和Western Blot方法检测HCV多CTL表位的表达.流式细胞术分析感染前后DC的CD80、CD83、CD86和人类白细胞抗原(HLA)-DR的表达,CCK-8法观察感染重组腺病毒的DC促进T细胞的增殖效应.ELISA检测重组腺病毒刺激后的DC培养上清液内白细胞介素(IL)-12及DC和T细胞混合培养上清液内干扰素(IFN)-γ的含量.用乳酸脱氢酶(LDH)释放法检测特异性CTL的杀伤活性.结果 成功构建含GFP标签的HCV多CTL表位的重组腺病毒,并在DC中表达.重组腺病毒能促进DC成熟,DC的CD80、CD83、CD86和HLA-DR的表达分别为(71.19±3.29)%、(81.21±5.07)%、(91.23±4.24)%、(97.95±5.31)%.感染DC后促进同源T细胞增殖,DC:T为1:10时增殖指数为6.806±0.247.分泌的IL-12和IFN-γ也明显增多,分别达到(193.83±6.25)pg/ml和(111.14±2.09)pg/ml.感染DC刺激的CTL能特异性杀伤转染FL-J6/JFH的Huh-7.5细胞,当效靶比为100:1时,ADI-DC-L的杀伤率为35.99%.结论 重组多CTL表位腺病毒在体外能有效感染DC,促进了T细胞反应,为下一步抗HCV的DC疫苗研制打下基础.%Objective To construct dendritic cell (DC) vaccine expressing hepatitis C virus (HCV) multi-cytotoxic T lymphocyte (CTL) epitopes, which can stimulate T-cell response in vitro, providing certain information for immunization experiments in vivo for the next step. Methods DCs were infected with recombinant defective adenoviruses expressing two HCV CTL epitopes with tagged green fluorescent protein (GFP). The rate of infection was detected by the fluorescence microscope or flow cytometry; the expression of multiple CTL epitopes in DC was proved by RT-PCR and Western Blot. The cell surface markers of DC such as CD80, CD83,CD86 and HLA-DR were identified by flow cytometry. T cell proliferation effect promoted by DC was observed by cell counting kit8(CCK-8). IL-12p70 in DC culture supernatant and IFN-γ in T cell supernatant were detected by Enzyme-Linked Immuno Sorbent Assay (ELISA). HCV specific CTL activity was measured by LDH release assay. Results Recombinant muiti-CTL epitopes and GFP were successfully expressed in DC. Adenovirus can promote DC maturation, the percentages of CDS0, CD83, CD86 and HLADR were (71.19±3.29)％, (81.21±5.07)％, (91.23±4.24)％, (97.95±5.31 )％ respectively. Infected DC promotes homologous T cell proliferation and the stimulation index was 6.806±0.247 when DC:T was 1:10. The secretion of IL-12 and IFN-γ was also increased to (193.83±6.25) pg/ml and (111.14±2.09) pg/ml respectively. The CTL stimulated by infected DC could specifically kill Huh-7.5 cells transfected with FL-J6/JFH transcripts. The cytotoxicity was 35.99％ when the rate of effector to target was 100:1. Conclusion The muiti-CTL epitopes recombinant adenovirus can effectively infect DC in vitro, promoting the T cell immune response, laying the foundation for developing anti-HCV DC vaccine.
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