冻融抗原负载的DC-CIK细胞对SKOV3的杀伤作用
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摘要
目的：探讨负载人卵巢癌细胞株SKOV3冻融抗原(Ag)的树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养后,对DC及CIK细胞增殖的影响；探讨与负载人卵巢癌细胞株SKOV3或COC1冻融Ag的DC共培养的CIK细胞对SKOV3杀伤作用的影响。
方法：选择12例上皮性卵巢癌患者,分离其外周血,获得单个核细胞(PBMC),用相应的诱导因子体外诱导出DC与CIK细胞。
分别收集处于对数生长期的SKOV3及COC1细胞,用细胞冻融法提取Ag,并于DC培养的第5天将Ag加入DC中培养,使之成为负载Ag的DC。将经Ag负载的DC与未经Ag负载的DC分别和CIK细胞共培养后分组：实验组：将经Ag负载的DC与CIK细胞共培养作为实验组(SKOV3Ag-DC+CIK组、COC1 Ag-DC+CIK组)。对照组：(1)将未经Ag负载的DC与CIK共培养作为对照组1(DC+CIK组)；(2)CIK细胞单独培养作为对照组2(CIK组)；(3)DC单独培养作为对照组3(DC组)；(4)负载抗原的DC为对照组4(Ag-DC组)。
自培养第1天到第20天,用台盼兰拒染法动态监测CIK细胞的增殖情况,观察DC及Ag负载的DC对CIK细胞增殖的影响。
用流式细胞技术分析DC组、SKOV3Ag-DC组、SKOV3Ag-DC-CIK组中DC细胞的表型,观察负载Ag及CIK对其增殖的影响。分析CIK组、DC-CIK组、SKOV3Ag-DC-CIK组中CIK细胞表型,观察DC及Ag-DC对CIK细胞增殖的影响。
用乳酸脱氢酶释放法检测CIK组、DC-CIK组、Ag-DC-CIK组(包括SKOV3-DC-CIK及COC1 -DC-CIK组)对SKOV3的杀伤活性,对比观察DC、SKOV3Ag-DC及COC1Ag -DC对CIK细胞杀伤活性的影响。
结果：与DC共培养后的CIK细胞的增殖速率大于单CIK细胞,但与同负载抗原的DC共培养的CIK细胞增殖率相比,差异无统计学意义(P>0.05)；Ag-DC-CIK组中DC细胞成熟表型高于DC组及Ag-DC组(P<0.01)；Ag-DC-CIK组中CIK细胞成熟表型高于CIK组及DC-CIK组(P<0.01)；SKOV3-DC-CIK组对SKOV3杀伤率高于CIK组、DC-CIK组及COC1-DC-CIK组(P<0.01)。
结论：(1)DC及负载SKOV3冻融Ag的DC与CIK共培养可促进DC、CIK细胞的成熟,但负载SKOV3冻融Ag的DC与DC相比,不能明显提高CIK细胞的增殖率；
(2)负载SKOV3冻融Ag的DC可增强CIK细胞对SKOV3的特异性杀伤作用。
Objective:To investigate the effect of dendritic cells(DC) pulsed with tumor lysate antigens of human ovarian cancer cell line SKOV3 co-cultured with cytokine induced killer(CIK) cells on the proliferation of DC and CIK cells and to investigate the impact on the cytotoxic activity against SKOV3 of CIK cells after its co-cultureding with DC that pulsed with tumor lysate antigens of SKOV3 or COC1.
Methods:Peripheral blood mononuclear cells(PBMC) isolated from 12 patients with ovarian cancer were induced to obtain CIK cells and DC respectively with the corresponding in vitro-inducible factors.
The Ag of SKOV3 and COC1 extracted using freeze-thaw method at the logarithmic growth phase were co-cultured with DC at the fifth day of its culture.Experiments included the experimental group and control group. CIK co-cultured with DC which were pulsed with tumor lysate antigens of SKOV3 were used as experimental group.(1)CIK co-cultured with DC without tumor antigens used as control group 1 (2) CIK cultured alone were used as control group 2.(3) DC cultured alone were used as control group 3. (4)DC pulsed with tumor lysate antigens of SKOV3 and COC1 were used as control group 4.
Trypan blue staining was used to observe the proliferation of CIK cells and the impaction of DC and DC pulsed with tumor lysate antigens on CIK cells since the 1 st training day to the 20th day.
The surface molecule expression of DC in DC group, Ag-DC group and Ag-DC-CIK group were measured by flow cytometry,and the impaction of Ag and CIK on DC was observed.
The surface molecule expression of CIK in CIK group, DC-CIK group and Ag-DC-CIK group were also measured by flow cytometry,and the impaction of DC and Ag-DC on CIK was observed too; The cytotoxic activities of CIK group, DC-CIK group and Ag-DC-CIK group against SKOV3 were measured using lactate dehydrogenase(LDH) assay,and the impaction of DC, SKOV3Ag-DC and COC1Ag-DC on the killing activity of CIK were observed contrastly.
Results:The proliferation rate of CIK cells co-cultured with DC was higher than the single CIK cells, while there was no statistical significance between the proliferation rate of CIK cells co-cultured with DC and the one of CIK cells co-cultured with antigen load DC(P>0.05). Compared with DC group and Ag-DC group, Ag-DC-CIK group led to a significant increase of mature phenotypes of DC(P<0.01); While the mature phenotypes of CIK in Ag-DC-CIK group also increased(P<0.01); The cytotoxic activities of SKOV3-DC-CIK group against SKOV3 cells were much higher than that of both CIK group, DC-CIK group and SKOV3-DC-CIK group (P<0.01).
Conclusions:(1)This study indicates that DC pulsed with tumor lysate antigens of SKOV3 co-cultured with CIK cells can promote the maturity of both DC and CIK. Compared with DC, DC pulsed with tumor lysate antigens of SKOV3 can not raise the growth rate of CIK cells;
(2)DC pulsed with tumor lysate antigens of SKOV3 co-cultured can improve the specific cytotoxic activity of CIK cells against SKOV3.
方法：选择12例上皮性卵巢癌患者,分离其外周血,获得单个核细胞(PBMC),用相应的诱导因子体外诱导出DC与CIK细胞。
分别收集处于对数生长期的SKOV3及COC1细胞,用细胞冻融法提取Ag,并于DC培养的第5天将Ag加入DC中培养,使之成为负载Ag的DC。将经Ag负载的DC与未经Ag负载的DC分别和CIK细胞共培养后分组：实验组：将经Ag负载的DC与CIK细胞共培养作为实验组(SKOV3Ag-DC+CIK组、COC1 Ag-DC+CIK组)。对照组：(1)将未经Ag负载的DC与CIK共培养作为对照组1(DC+CIK组)；(2)CIK细胞单独培养作为对照组2(CIK组)；(3)DC单独培养作为对照组3(DC组)；(4)负载抗原的DC为对照组4(Ag-DC组)。
自培养第1天到第20天,用台盼兰拒染法动态监测CIK细胞的增殖情况,观察DC及Ag负载的DC对CIK细胞增殖的影响。
用流式细胞技术分析DC组、SKOV3Ag-DC组、SKOV3Ag-DC-CIK组中DC细胞的表型,观察负载Ag及CIK对其增殖的影响。分析CIK组、DC-CIK组、SKOV3Ag-DC-CIK组中CIK细胞表型,观察DC及Ag-DC对CIK细胞增殖的影响。
用乳酸脱氢酶释放法检测CIK组、DC-CIK组、Ag-DC-CIK组(包括SKOV3-DC-CIK及COC1 -DC-CIK组)对SKOV3的杀伤活性,对比观察DC、SKOV3Ag-DC及COC1Ag -DC对CIK细胞杀伤活性的影响。
结果：与DC共培养后的CIK细胞的增殖速率大于单CIK细胞,但与同负载抗原的DC共培养的CIK细胞增殖率相比,差异无统计学意义(P>0.05)；Ag-DC-CIK组中DC细胞成熟表型高于DC组及Ag-DC组(P<0.01)；Ag-DC-CIK组中CIK细胞成熟表型高于CIK组及DC-CIK组(P<0.01)；SKOV3-DC-CIK组对SKOV3杀伤率高于CIK组、DC-CIK组及COC1-DC-CIK组(P<0.01)。
结论：(1)DC及负载SKOV3冻融Ag的DC与CIK共培养可促进DC、CIK细胞的成熟,但负载SKOV3冻融Ag的DC与DC相比,不能明显提高CIK细胞的增殖率；
(2)负载SKOV3冻融Ag的DC可增强CIK细胞对SKOV3的特异性杀伤作用。
Objective:To investigate the effect of dendritic cells(DC) pulsed with tumor lysate antigens of human ovarian cancer cell line SKOV3 co-cultured with cytokine induced killer(CIK) cells on the proliferation of DC and CIK cells and to investigate the impact on the cytotoxic activity against SKOV3 of CIK cells after its co-cultureding with DC that pulsed with tumor lysate antigens of SKOV3 or COC1.
Methods:Peripheral blood mononuclear cells(PBMC) isolated from 12 patients with ovarian cancer were induced to obtain CIK cells and DC respectively with the corresponding in vitro-inducible factors.
The Ag of SKOV3 and COC1 extracted using freeze-thaw method at the logarithmic growth phase were co-cultured with DC at the fifth day of its culture.Experiments included the experimental group and control group. CIK co-cultured with DC which were pulsed with tumor lysate antigens of SKOV3 were used as experimental group.(1)CIK co-cultured with DC without tumor antigens used as control group 1 (2) CIK cultured alone were used as control group 2.(3) DC cultured alone were used as control group 3. (4)DC pulsed with tumor lysate antigens of SKOV3 and COC1 were used as control group 4.
Trypan blue staining was used to observe the proliferation of CIK cells and the impaction of DC and DC pulsed with tumor lysate antigens on CIK cells since the 1 st training day to the 20th day.
The surface molecule expression of DC in DC group, Ag-DC group and Ag-DC-CIK group were measured by flow cytometry,and the impaction of Ag and CIK on DC was observed.
The surface molecule expression of CIK in CIK group, DC-CIK group and Ag-DC-CIK group were also measured by flow cytometry,and the impaction of DC and Ag-DC on CIK was observed too; The cytotoxic activities of CIK group, DC-CIK group and Ag-DC-CIK group against SKOV3 were measured using lactate dehydrogenase(LDH) assay,and the impaction of DC, SKOV3Ag-DC and COC1Ag-DC on the killing activity of CIK were observed contrastly.
Results:The proliferation rate of CIK cells co-cultured with DC was higher than the single CIK cells, while there was no statistical significance between the proliferation rate of CIK cells co-cultured with DC and the one of CIK cells co-cultured with antigen load DC(P>0.05). Compared with DC group and Ag-DC group, Ag-DC-CIK group led to a significant increase of mature phenotypes of DC(P<0.01); While the mature phenotypes of CIK in Ag-DC-CIK group also increased(P<0.01); The cytotoxic activities of SKOV3-DC-CIK group against SKOV3 cells were much higher than that of both CIK group, DC-CIK group and SKOV3-DC-CIK group (P<0.01).
Conclusions:(1)This study indicates that DC pulsed with tumor lysate antigens of SKOV3 co-cultured with CIK cells can promote the maturity of both DC and CIK. Compared with DC, DC pulsed with tumor lysate antigens of SKOV3 can not raise the growth rate of CIK cells;
(2)DC pulsed with tumor lysate antigens of SKOV3 co-cultured can improve the specific cytotoxic activity of CIK cells against SKOV3.
引文
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