CpG ODN增强树突状细胞对胃癌细胞增殖周期和凋亡影响的实验研究

Study on gastric cancer cell proliferation cycle and apoptosis with dendritic cells induced by CpG ODN1826

目的探讨CpG ODN1826增强树突状细胞(DC)抗胃癌效应的作用。方法分离正常人外周血DC,用GM-CSF和IL-4培养,于第5天加入TNF-α并分成Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅷ组继续培养。第6天各组均加入胃癌细胞冻融抗原50μl,Ⅱ、Ⅳ、Ⅵ、Ⅷ组再分别加入CpG ODN182610μg/ml,第10天ELISA检测IL-12和IFN-γ的水平,MTT法检测CTL对MKN45、MKN28、SGC7901和A549细胞的体外杀伤效应。流式细胞术检测CpG ODN1826增强DC对胃癌细胞增殖周期、凋亡的影响。结果体外培养第10天,加入CpG ODN1826后各组IL-12、IFN-γ的分泌量明显提高;CpG ODN1826协助DC对同种不同分化类型的胃癌细胞株MKN45、MKN28、SGC7901均有强烈的杀伤效应(P<0.01),杀伤活性显著高于A549(P<0.01)。体外应用CpG ODN1826对肿瘤细胞周期比例:G0/G1间期(MKN4568.35%、MKN2869.23%、SGC790169.80%),S期(MKN4539.45%、MKN2839.75%、SGC790139.55%),G2/M期(MKN456.50%、MKN286.30%、SGC79016.42%);与A549(G0/G1间期57.68%,S期25.13%,G2/M期18.46%)比较,差异有统计学意义(P<0.01);同时,不同肿瘤细胞株的凋亡率分别为MKN4575.20%、MKN2373.87%、SGC790172.43%、A54951.43%,表明CpG ODN1826能显著增强DC对胃癌细胞周期的抑制作用,促进胃癌细胞的早期凋亡(P<0.01)。结论 CpG ODN1826体外可显著增强DC诱导出高效而特异的抗胃癌效应,显著抑制胃癌细胞分裂增殖、促进凋亡,可作为胃癌免疫治疗的一种有效方法。

Objective To explore whether CpG ODN1826 assist dendritic cells（DC） to affect the anti-gastric cancer effects in vitro.Methods DC was isolated from normal human peripheral blood by GM-CSF＋IL-4,and append TNF-α in the fifth and divided Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,Ⅶ,Ⅷ groups,and append carcinoma antigen 50 μl in every group,CpG ODN1826 10 μg/ml in Ⅱ,Ⅴ,Ⅵ,Ⅷ groups in the sixth day.IL-12 and IFN-γ levels were detected using ELISA in 10th day.The cytotoxicity of CTL to MKN45,MKN28,SGC7901,A549 was detected by MTT assay in vitro.The inhibitive effects of CpG ODN1826 on gastric cell proliferation cycle and apoptosis in vitro were detected by flow cytometry.Results On the 10th day,IL-12,IFN-γ level were significantly increased in the CpG ODN1826 groups.The CTL had much obviously raised the cytotoxicity to different differentiated types of the three gastric cancer cell lines such MKN45,MKN28,SGC7901 by CpG ODN1826,and than A549 （P0.01）.Treated with the CpG ODN1826 in vitro,the percentages of G0/G1,S and G2/M cells of tumor cells were（MKN45 68.35%,MKN28 69.23%,SGC790169.80%）,（MKN45 39.45%,MKN28 39.75%,SGC7901 39.55%） and（MKN45 6.50%,MKN28 6.30%,SGC7901 6.42%） respectively.and the apoptosis of MKN45 75.20%,MKN23 73.87%,SGC7901 72.43%,A549 51.43%,indicated CpG ODN1826 may significantly inhibit the percentages of three gastric cell lines,and promote gastric cell early apoptosis（P0.01）.Conclusions The effecience and specificity of CpG ODN1826 can observably enhance dendritic cells induce the effecience and specificity of anti-gastric cancer activity,can significantly inhibit gastric cells proliferation and growth,and promote gastric cell early apoptosis,and might provide a new kind of therapeutic means for gastric cancer.