人天冬氨酸-β-羟化酶树突状细胞疫苗对肝癌细胞系的体外作用研究

Function of dendritic cells vaccines targeting aspartate-β-hydroxylase on hepatocellular carcinoma in vitro

摘要:

目的：通过对携带人天冬氨酸-β-羟化酶（aspartate-β-hydroxylase，AAH）基因的树突状细胞杀伤肝癌细胞系的研究，探索可用于防治肝癌的树突状细胞疫苗。方法：重组腺病毒（Ad-AAH-IRES2-EGFP）感染C57BL/6小鼠骨髓未成熟树突状细胞（dendritic cells，DCs）制备携带AAH基因的DCs（AAH-DCs），与同体小鼠脾脏T淋巴细胞共培养后作为效应细胞，以肝癌细胞株HepG2和Huh7分别作为靶细胞，效应细胞分别和靶细胞共培养24 h，CCK-8法检测效应细胞对靶细胞增殖的影响；流式细胞术检测效应细胞对靶细胞凋亡的影响。将AAH-DCs连续3次皮下注射免疫C57BL/6小鼠，乳酸脱氢酶释放法检测免疫后小鼠脾脏T淋巴细胞对靶细胞的裂解效应。结果：AAH-DCs与T淋巴细胞共培养组靶细胞的增殖能力明显低于对照组（P=0.000），凋亡率明显高于对照组（P=0.000）；实验组靶细胞的裂解率明显高于对照组（P=0.000）。结论：荷载AAH基因的树突状细胞疫苗能够诱导产生细胞毒性T淋巴细胞从而杀伤表达AAH的肝癌细胞。

Abstract:

Objective：To explore the antitumor effect of dendritic cells（DCs） vaccine targeting aspartate-β-hydroxylase（AAH） gene against hepatocellular carcinoma（HCC） cells HepG2 and Huh7 for searching DCs vaccine for hepatocarcinoma. Methods：AAH-DCs vaccine was prepared in vitro via infecting DCs with recombinant adenovirus vector encoding AAH. T cells co-cultured with AAH-DCs vaccine were served as the effector cells and human HCC cell lines HepG2 and Huh7 were regarded as the target cells. The prolifer－ation and apoptosis of target cells，after co-culturing with the effector cells for 24h，were detected by CCK-8 and FCM analysis re－spectively. C57BL/6 mice were immunized with AAH-DCs vaccines by three continuous subcutaneous injections，and the cytotoxic effect of T cells against target cells was observed by lactate dehydrogenase（LDH） release assay. Results：Compared with the control groups，the declined proliferation activity of target cells in AAH-DCs vaccine group was found by CCK-8，while increased apoptosis rate of target cells in AAH-DCs vaccine group was tested by FCM（P=0.000）. Additionally，LDH release assay showed that the cytolysis effect of target cells in AAH-DCs vaccine group was higher than that in other control groups（P=0.000）. Conclusion：DCs vaccines targeting AAH can induce the production of cytotoxicity T lymphocytes for fulfilling the killing of HCC cells expressing AAH gene.