肿瘤抗原负载的DC-CIK细胞增殖能力及对肝癌细胞HepG2的杀伤活性研究

Study of proliferation ability of tumor antigen-loaded DC-CIK cells and its killing effect on hepatocarcinoma cells HepG2

目的观察肿瘤抗原负载的树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养后的增殖能力及对肝癌细胞HepG2的杀伤活性。方法采用液氮反复冻融法制备肝癌细胞HepG2抗原。应用血细胞分离机采集健康供血者单个核细胞,诱导并分离出DC、CIK,用肿瘤抗原冲击DC获得Ag-DC,将CIK分为3组,一组为单纯CIK,二组以DC∶CIK=1∶5比例混合,三组以Ag-DC∶CIK=1∶5比例混合。3组细胞分别记作:CIK组、DC-CIK组和Ag-DC-CIK组。观察3组细胞的增殖能力和细胞表型,并用四甲基偶氮唑蓝(MTT)法测定3组细胞对肝癌细胞HepG2的杀伤活性。结果3组细胞随着培养时间的延长增殖速度逐渐增快,在共同培养的第9天、第12天和第15天时Ag-DC-CIK的增殖倍数(61.32±1.72、190.83±3.53、399.09±5.60)均明显高于同时间CIK的增殖倍数(22.47±2.07、55.91±1.81、83.20±2.34)和DC-CIK的增殖倍数(40.26±2.49、125.03±4.16、251.55±3.25),3组间差异有统计学意义(F=185.78,P=0.033;F=297.35,P=0.018;F=455.37,P<0.001),进一步两两比较发现,差异均具有统计学意义(均P<0.05)。Ag-DC-CIK对HepG2细胞的杀伤活性在效靶比为5∶1、10∶1和20∶1时分别为31.71%±0.29%、42.43%±1.86%和57.69%±1.11%,均显著高于CIK组(12.11%±1.14%、21.30%±0.52%和30.71%±1.26%)和DC-CIK组(20.06%±0.67%、29.89%±1.37%和39.11%±0.92%),3组间差异有统计学意义(F=159.64,P=0.037;F=199.36,P=0.025;F=302.08,P<0.001),进一步两两比较发现,差异均具有统计学意义(均P<0.05)。CIK、DC-CIK和Ag-DC-CIK 3组细胞培养至第15天,流式细胞仪分析表明3组细胞均表达CD3^+CD8^+、CD3^+CD56^+双阳性细胞,且Ag-DC-CIK组CD3^+CD8^+、CD3^+CD56^+双阳性细胞的含量(88.12%±1.24%、61.35%±2.63%)明显高于CIK组(54.37%±3.08%、18.22%±1.83%)和DC-CIK组(69.80%±1.46%、39.51%±2.17%),3组间差异有统计学意义(F=414.32,P<0.001;F=378.60,P<0.001),进一步两两比较发现,差异均具有统计学意义(均P<0.001)。结论肿瘤抗原负载DC后与CIK共培养获得的Ag-DC-CIK细胞的增殖能力和对肝癌细胞HepG2的杀伤活性显著高于CIK细胞和DC-CIK细胞。

ObjectiveTo observe the proliferation ability of cocultured dendritic cells(DCs)loaded with tumor antigen and cytokine-induced killer cells(CIKs)and its killing effect on hepatocarcinoma cells HepG2.MethodsThe antigen of hepatocarcinoma cells HepG2 was prepared by repeated freezing and thawing of liquid nitrogen.Peripheral blood mononuclear cells(PBMNCs)were isolated from healthy donors by blood cell separator,then DCs and CIKs were induced.Ag-DCs were obtained by impinging DCs with tumor antigens.CIKs were divided into three groups:the first group was CIKs alone,the second group was mixed in the proportion of DCs∶CIKs=1∶5,and the third group was mixed in the proportion of Ag-DCs∶CIKs=1∶5.The three groups of cells were recorded as CIK group,DC-CIK group and Ag-DC-CIK group.The proliferation and cell phenotype of the three groups of cells were observed and the killing effects on hepatocarcinoma cells HepG2 were detected by methyl thiazolyl tetrazolium(MTT)assay.ResultsThe proliferation multiples of the three groups of cells were gradually increased with the prolongation of culture time,and the proliferation rates of Ag-DC-CIK on the 9th day(61.32±1.72),the 12th day(190.83±3.53)and the 15th day(399.09±5.60)were significantly higher than those of CIK group(22.47±2.07,55.91±1.81,83.20±2.34)and DC-CIK group(40.26±2.49,125.03±4.16,251.55±3.25),and the difference between the three group was statistically significant(F=185.78,P=0.033;F=297.35,P=0.018;F=455.37,P<0.001),in addition,the differences between each two groups were statistically significant(all P<0.05).The cytotoxicity of Ag-DC-CIK to HepG2 cells at the effective target ratios of 5∶1(31.71%±0.29%),10∶1(42.43%±1.86%)and 20∶1(57.69%±1.11%)were significantly higher than those of CIK group(12.11%±1.14%,21.30%±0.52%,30.71%±1.26%)and DC-CIK group(20.06%±0.67%,29.89%±1.37%,39.11%±0.92%),and the difference between the three group was statistically significant(F=159.64,P=0.037;F=199.36,P=0.025;F=302.08,P<0.001),in addition,the differences between each two groups were statistically significant(all P<0.05).On the 15th day of cell culture,the flow cytometry analysis showed that all the three groups were expressed CD3^+CD8^+,CD3^+CD56^+double positive cells,the contents of CD3^+CD8^+、CD3^+CD56^+double positive cells in the Ag-DC-CIK group(88.12%±1.24%,61.35%±2.63%)were significantly higher than those in the CIK group(54.37%±3.08%,18.22%±1.83%)and DC-CIK group(69.80%±1.46%,39.51%±2.17%),and the difference between the three group was statistically significant(F=414.32,P<0.001;F=378.60,P<0.001),in addition,the differences between each two groups were statistically significant(all P<0.001).ConclusionThe proliferation ability and killing effect of Ag-DC-CIK that obtained from antigen-pulsed DCs co-cultured with CIKs are significantly higher than those of CIKs and DC-CIKs.