人ASPH基因修饰树突状细胞介导CTLs对肝癌细胞系SMMC-7721的体外杀伤作用

Killing effect of cytotoxic T lymphocytes induced by dendritic cells transfected with human aspartate-β-hydroxylase gene on hepatocellular carcinoma cells line SMMC-7721 in vitro

目的:探索人天冬氨酸-β-羟化酶(aspartateβ-hydroxylase,ASPH)基因修饰的树突状细胞(dendritic cells,DCs)诱导细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)对肝癌细胞系SMMC-7721的体外杀伤效应。方法:重组腺病毒(Ad-ASPHIRES2-GFP)感染C57BL/6小鼠骨髓未成熟树突状细胞(immature DCs,im DCs)制备DCs疫苗,分为3组:含人ASPH基因的重组腺病毒感染im DCs组(DCs-ASPH组)、空腺病毒感染im DCs组(DCs-GFP组)和im DCs组,Western blot和q RT-PCR检测ASPH蛋白和m RNA在各组DCs中的表达,流式细胞术(flow cytometry,FCM)检测感染前后小鼠DCs表面标志物CD80、主要组织相容性复合物(major histocompatibility complex-Ⅱ,MHC-Ⅱ)和CD11c表达量。DCs疫苗诱导产生CTLs后与肝癌细胞系SMMC-7721共培养,采用CCK-8法和FCM分别检测其对SMMC-7721细胞增殖和凋亡的影响。ELISA检测CTLs上清液中IFN-γ含量。结果:成功培养出DCs并制备DCs疫苗,人ASPH基因修饰的DCs表面标志物CD80、MHC-Ⅱ和CD11c显著高于DCs-GFP组(P=0.000)和im DCs组(P=0.000)。人ASPH基因修饰的DCs诱导的CTLs组SMMC-7721细胞的增殖能力显著低于DCs-GFP组和im DCs组(P=0.000)、凋亡率明显高于DCs-GFP组和im DCs组(P=0.000),DCs-ASPH组诱导的CTLs上清液中IFN-γ显著高于DCs-GFP组和im DCs组(P=0.000)。结论:人ASPH基因修饰的DCs疫苗体外诱导产生的CTLs能够明显抑制肝癌细胞SMMC-7721增殖并促进其凋亡。

Objective：To investigate the killing effect of cytotoxic T lymphocytes（CTLs）on hepatocellular carcinoma cells line SMMC-7721 after being induced by dendritic cells（DCs）transfected with human aspartate-β-hydroxylase gene in vitro. Methods：Immature dendritic cells,from the bone marrow of the mice C57BL/6,loaded with human aspartate-β-hydroxylase recombinant adenovirus were used to prepare for DCs vaccine. They were classified as immature dendritic cells infected by recombinant adenovirus loaded with human aspartate-β-hydroxylase（DCs-ASPH group）,immature dendritic cells infected by recombinant adenovirus（DCs-GFP group）and immature dendritic cells group（im DCs group）. The expressions of ASPH protein and m RNA in each DCs were detected by Western blot and q RT-PCR,and the surface expression of CD80,MHC-Ⅱ and CD11 c were examined by flow cytometry（FCM）in both uninfected and infected dendritic cells. The specific CTLs induced by DCs vaccine were subsequently co-cultured with hepatocellular carcinoma cells line SMMC-7721. The proliferation and apoptosis rate of SMMC-7721 cells were detected by cell counting kit 8（CCK-8）and flow cytometry. The level of IFN-γ was measured by enzyme-linked immunosorbent assay（ELISA）. Results：DCs was successfully cultured and DCs vaccine was constructed. The DCs loaded with human aspartate-β-hydroxylase recombinant adenovirus expressed significantly higher level of CD80（P=0.000）,MHC-Ⅱ（P=0.000）and CD11c（P=0.000）than those in DCsGFP group and im DCs group. The proliferation of SMMC-7721 cells co-cultured with CTLs induced by human aspartate-β-hydroxylase recombinant adenovirus was lower while the apoptosis rate was higher than those in DCs-GFP group and im DCs group（P=0.000）.The secretion level of IFN-γ in CTLs induced by DCs-ASPH was also significantly higher than that in DCs-GFP group and im DCs group（P=0.000）. Conclusion：CTLs induced by DCs transfected with human aspartate-β-hydroxylase recombinant adenovirus can apparently inhibit the proliferation of hepatocellular carcinoma cells line SMMC-7721,and induce cells apoptosis in vitro.