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目的:制备来源于人外周血并转染人乳头瘤病毒(HPV)16E6基因的冻融树突状细胞(DC)疫苗,检测其细胞形态,转染情况及体外诱导的免疫效应。方法:细胞因子扩增人外周血DC,低温-70℃冻存;Lipofectamine转染HPV16E6制备DC疫苗。动态形态学观察,免疫细胞化学检测E6分子表达,体外诱导并测定细胞毒性T淋巴细胞活性。结果:转染E6基因的人外周血冻融DC呈形态迥异的多突起状,免疫细胞化学染色显示E6蛋白表达阳性;MTT法检测人宫颈癌细胞株(CaSki细胞)的杀伤活性明显高于对照组(P<0.01),与新鲜DC疫苗比无统计学意义(P>0.05)。结论:转染E6基因的人外周血冻融DC疫苗保持了功能成熟DC的形态特征,且内源性表达E6蛋白,并在体外诱导出高效特异性的抗宫颈癌免疫反应,为宫颈癌的主动特异性免疫治疗提供了理想的疫苗和实验依据。
OBJECTIVE: To prepare a freeze-thaw dendritic cell (DC) vaccine derived from human peripheral blood and transfected with human papillomavirus (HPV) 16E6 gene. The cell morphology, transfection and in vitro immune response were detected. Methods: Human peripheral blood DCs were expanded by cytokines and cryopreserved at -70 ℃. DC vaccine was transfected by HPV16E6 by Lipofectamine. Dynamic morphological observation, immunocytochemistry detection of E6 molecule expression in vitro induction and determination of cytotoxic T lymphocyte activity. Results: Frozen-thawed DCs from human peripheral blood transfected with E6 gene showed multi-protuberances with different morphologies, and immunocytochemical staining showed positive expression of E6 protein. The cytotoxic activity of E6 gene in human cervical cancer cell lines (CaSki cells) was significantly higher than that of the control Group (P <0.01), compared with the fresh DC vaccine was not statistically significant (P> 0.05). CONCLUSION: The human peripheral blood freeze-thaw DC vaccine transfected with E6 gene retains the morphological characteristics of mature DCs and endogenously expresses E6 protein and induces highly specific and specific anti-cervical cancer immune responses in vitro for cervical cancer Proactive immunotherapy provides the ideal vaccine and experimental evidence.