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目的:采用人乳头状瘤-16(HPV-16)E6E7重组腺病毒(pAd-E6E7)转染树突状细胞(Dendritic cell,DC),观察基因修饰的DC疫苗诱导细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte,CTL)致使CaSki细胞凋亡的效果。方法:将pAd-E6E7转染体外培养的小鼠未成熟树突状细胞制备DC疫苗,激光共聚焦显微镜观察转染的小鼠未成熟树突状细胞绿色荧光蛋白表达,流式细胞术检测转染前后小鼠树突状细胞表面标志物(CD40、CD86、MHCⅡ和CD11C)。DC疫苗诱导产生特异性细胞毒性T淋巴细胞,与CaSki细胞共培养后,采用DAPI、TUNEL及流式细胞术检测CaSki细胞凋亡情况。结果:pAd-E6E7成功转染体外培养的小鼠未成熟树突状细胞,体外转染效率约为40%～50%,成功制备了HPV16 E6E7基因修饰树突状细胞疫苗,诱导产生细胞毒性T淋巴细胞,经DAPI、TUNEL及流式细胞术检测证明CaSki出现凋亡。结论:以带有HPV16 E6E7基因的重组腺病毒载体转染DC制备基因修饰的DC疫苗,诱导CTL致使CaSki细胞出现凋亡。
Objective: To investigate the effect of gene-modified DC vaccine on cytotoxic T lymphocytes (Cytotoxicity) by transfecting dendritic cells (DCs) with human papilloma-16 (HPV16) E6E7 recombinant adenovirus (pAd-E6E7) Tlymphocyte, CTL) induced apoptosis in CaSki cells. Methods: DC vaccine was prepared from immature dendritic cells transfected with pAd-E6E7 in vitro. The expression of green fluorescent protein (EGFP) in immature dendritic cells was detected by laser scanning confocal microscopy. Flow cytometry Dendritic cell surface markers (CD40, CD86, MHC II, and CD11C) before and after staining. DC vaccine induced specific cytotoxic T lymphocytes, co-cultured with CaSki cells, the use of DAPI, TUNEL and flow cytometry CaSki cell apoptosis. Results: pAd-E6E7 was successfully transfected into mouse immature dendritic cells in vitro and the transfection efficiency was about 40% -50% in vitro. HPV16 E6E7 gene-modified dendritic cell vaccine was successfully prepared to induce cytotoxic T Lymphocytes were detected by DAPI, TUNEL and flow cytometry CaSki apoptosis. CONCLUSION: Gene-modified DC vaccines were transfected with recombinant adenovirus vector carrying HPV16 E6E7 gene and induce CTL induced apoptosis in CaSki cells.