树突状细胞疫苗的制备及其体外诱导细胞毒性T淋巴细胞对宫颈癌CaSki细胞的杀伤作用

Induction of cytotoxic T lymphocytes against cervical cancer cell line CaSki using therapeutic dendritic cell vaccine

目的:制备树突状细胞(dendritic cells,DCs)疫苗并观察其在体外诱导细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)对宫颈癌CaSki细胞的杀伤效应。方法:分离培养小鼠未成熟DCs,FCM检测小鼠未成熟DCs表面标志物CD40、CD86、主要组织相容性复合体-Ⅱ(major histocompatibility complex-Ⅱ,MHC-Ⅱ)和CD11c;将已成功构建的携带人乳头状瘤病毒(human papillomavirus,HPV)16E6E7基因的重组腺病毒pAd-E6E7感染体外培养的小鼠未成熟DCs,提取CaSki细胞裂解物负载DCs,制备DC疫苗,激光共聚焦显微镜下观察pAd-E6E7感染的小鼠未成熟DCs绿色荧光蛋白表达,Western印迹法检测E6蛋白的表达;DCs疫苗诱导产生CTLs后与CaSki细胞共培养,CCK8(cell countingkit8)法检测其对CaSki细胞的杀伤效应。结果:成功分离培养了小鼠未成熟DCs,并制备获得了HPV16E6E7特异性DCs疫苗。DCs疫苗诱导产生的CTLs对CaSki细胞具有杀伤作用,pAd-E6E7感染组对CaSki的杀伤效应明显高于CaSki细胞裂解物负载组及未处理DCs组(P<0.05)。结论:以携带HPV16E6E7基因的重组腺病毒感染DCs制备基因修饰的DCs疫苗,具有诱导CTLs体外杀伤子宫颈癌CaSki细胞的作用。

Objective：To observe the inductive effect of cytotoxic T lymphocytes（CTLs） against human cervical cancer cell line CaSki using therapeutic dendritic cells（DCs） vaccine in vitro.Methods：Immature mouse DCs were isolated and cultured.The expressions of cell-surface CD40,CD86,major histocompatibility complex（MHC）-Ⅱ and CD11c in immature DCs were detected by flow cytometry（FCM）.Then the immature mouse DCs were infected with recombinant adenoviral vector carrying human papillomavirus（HPV）16 E6/E7（pAd-E6/E7）,and the CaSki cell lysate-loaded autologous DCs vaccine was prepared.The expression of green fluorescent protein in pAd-E6/E7-infected immature mouse DCs was observed under a laser scanning confocal microscope,and the expression of E6 protein was detected by Western blotting.DCs vaccine was used to induce specific CTLs,were subsequently co-cultured with CaSki cells.The killing effect of CTLs against CaSki cells was determined using cell counting kit8（CCK8） assay.Results：HPV16 E6/E7-specific DCs vaccine was successfully prepared.CTLs which induced by DCs vaccine exerted a killing effect on CaSki cells.This killing effect was higher in pAd-E6/E7-infected group than those in CaSki cell lysate-loaded group and the untreated control group（P0.05）.Conclusion：Genetically modified DC vaccine can successfully be prepared by infection with pAd-E6/E7,and it has a significant effect on triggering of specific CTLs against CaSki cells.