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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 prepare dendritic cells (DCs) vaccines and observe the cytotoxicity of cytotoxic T lymphocytes (CTLs) on cervical cancer CaSki cells in vitro. Methods: Mouse immature DCs were isolated and cultured. The surface markers CD40, CD86, major histocompatibility complex-Ⅱ (MHC-Ⅱ) and CD11c in immature DCs were detected by FCM. The constructed recombinant adenovirus pAd-E6E7 carrying human papillomavirus (HPV) 16E6E7 gene was used to infect mouse immature DCs cultured in vitro and the CaSki cell lysate was loaded to load DCs to prepare a DC vaccine. Laser confocal microscopy The expression of green fluorescent protein (EGFP) of immature DCs in mice infected with pAd-E6E7 was observed, and the expression of E6 protein was detected by Western blotting. CTLs induced by DCs vaccine were co-cultured with CaSki cells and the killing activity of CaSki cells by CCK8 effect. Results: Mouse immature DCs were successfully isolated and cultured, and HPV16E6E7-specific DCs vaccine was obtained. The cytotoxicity of CTLs induced by DCs vaccine on CaSki cells was significantly lower than that of CaSki cells (P <0.05). CONCLUSION: DCs vaccinated with DCs carrying recombinant adenovirus carrying HPV16E6E7 gene have the potential of inducing CTLs to kill cervical cancer CaSki cells in vitro.