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。
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 proliferation and apoptosis of target cells,after co-culturing with the effector cells for 24 h,were detected by CCK-8 and FCM analysis respectively. C57 BL/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.
引文
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