冻融抗原负载的DC-CIK细胞对SKOV3的杀伤作用
发布时间：2019-06-04 00:45
【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(P0.05)；Ag-DC-CIK组中DC细胞成熟表型高于DC组及Ag-DC组(P0.01)；Ag-DC-CIK组中CIK细胞成熟表型高于CIK组及DC-CIK组(P0.01)；SKOV3-DC-CIK组对SKOV3杀伤率高于CIK组、DC-CIK组及COC1-DC-CIK组(P0.01)。 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。
[Abstract]:Objective: to investigate the effect of dendritic cell (DC) loaded with human ovarian cancer cell line SKOV3 freeze-thaw antigen (Ag) on the proliferation of DC and CIK cells after co-culture with cytokine-induced killer cell (CIK). To investigate the effect of CIK cells co-cultured with DC loaded with human ovarian cancer cell line SKOV3 or COC1 freeze-thawed Ag on the killing effect of SKOV3. Methods: the peripheral blood of 12 patients with epithelial ovarian cancer was isolated and mononuclear cells (PBMC), were obtained to induce DC and CIK cells with corresponding inducers in vitro. SKOV3 and COC1 cells in logarithmic growth phase were collected. Ag, was extracted by cell freeze-thaw method and Ag was added to DC on the 5th day of DC culture to make it DC. loaded with Ag. DC loaded with Ag and DC loaded with Ag were co-cultured with CIK cells: experimental group: DC loaded with Ag was co-cultured with CIK cells as experimental group (SKOV3Ag-DC CIK group, COC1 Ag-DC CIK group). Control group: (1) DC and CIK were co-cultured with CIK as control group 1 (DC CIK group); (2) CIK cells were cultured alone as control group 2 (CIK group); (3) DC as control group 3 (DC group); (4) the DC loaded with antigen was control group 4 (Ag-DC group). From the first day to the 20th day of culture, the proliferation of CIK cells was dynamically monitored by trypan blue exclusion method, and the effects of DC and Ag loaded DC on the proliferation of CIK cells were observed. The phenotypes of DC cells in DC group, SKOV3Ag-DC group and SKOV3Ag-DC-CIK group were analyzed by flow cytometry, and the effects of Ag and CIK on their proliferation were observed. The phenotypes of CIK cells in CIK group, DC-CIK group and SKOV3Ag-DC-CIK group were analyzed, and the effects of DC and Ag-DC on the proliferation of CIK cells were observed. The cytotoxicity of CIK group, DC-CIK group and Ag-DC-CIK group (including SKOV3-DC-CIK and COC1-DC-CIK group) to SKOV3 was detected by lactic dehydrogenase release assay. The effects of DC,SKOV3Ag-DC and COC1Ag-DC on the killing activity of CIK cells were compared. Results: the proliferation rate of CIK cells co-cultured with DC was higher than that of single CIK cells, but there was no significant difference compared with the proliferation rate of CIK cells co-cultured with DC loaded with antigen (P 0.05). The mature phenotype of DC cells in Ag-DC-CIK group was higher than that in DC group and Ag-DC group (P 0.01), and the maturation phenotype of CIK cells in Ag-DC-CIK group was higher than that in CIK group and DC-CIK group (P 0.01). The killing rate of SKOV3 in SKOV3-DC-CIK group was higher than that in CIK group, DC-CIK group and COC1-DC-CIK group (P 0.01). Conclusion: (1) Co-culture of DC and CIK loaded with SKOV3 freeze-thawed Ag can promote the maturation of DC,CIK cells, but DC loaded with SKOV3 freeze-thawed Ag can not significantly increase the proliferation rate of CIK cells compared with DC. (2) DC loaded with SKOV3 freeze-thawed Ag enhanced the specific killing effect of CIK cells on SKOV3.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2492349
[Abstract]:Objective: to investigate the effect of dendritic cell (DC) loaded with human ovarian cancer cell line SKOV3 freeze-thaw antigen (Ag) on the proliferation of DC and CIK cells after co-culture with cytokine-induced killer cell (CIK). To investigate the effect of CIK cells co-cultured with DC loaded with human ovarian cancer cell line SKOV3 or COC1 freeze-thawed Ag on the killing effect of SKOV3. Methods: the peripheral blood of 12 patients with epithelial ovarian cancer was isolated and mononuclear cells (PBMC), were obtained to induce DC and CIK cells with corresponding inducers in vitro. SKOV3 and COC1 cells in logarithmic growth phase were collected. Ag, was extracted by cell freeze-thaw method and Ag was added to DC on the 5th day of DC culture to make it DC. loaded with Ag. DC loaded with Ag and DC loaded with Ag were co-cultured with CIK cells: experimental group: DC loaded with Ag was co-cultured with CIK cells as experimental group (SKOV3Ag-DC CIK group, COC1 Ag-DC CIK group). Control group: (1) DC and CIK were co-cultured with CIK as control group 1 (DC CIK group); (2) CIK cells were cultured alone as control group 2 (CIK group); (3) DC as control group 3 (DC group); (4) the DC loaded with antigen was control group 4 (Ag-DC group). From the first day to the 20th day of culture, the proliferation of CIK cells was dynamically monitored by trypan blue exclusion method, and the effects of DC and Ag loaded DC on the proliferation of CIK cells were observed. The phenotypes of DC cells in DC group, SKOV3Ag-DC group and SKOV3Ag-DC-CIK group were analyzed by flow cytometry, and the effects of Ag and CIK on their proliferation were observed. The phenotypes of CIK cells in CIK group, DC-CIK group and SKOV3Ag-DC-CIK group were analyzed, and the effects of DC and Ag-DC on the proliferation of CIK cells were observed. The cytotoxicity of CIK group, DC-CIK group and Ag-DC-CIK group (including SKOV3-DC-CIK and COC1-DC-CIK group) to SKOV3 was detected by lactic dehydrogenase release assay. The effects of DC,SKOV3Ag-DC and COC1Ag-DC on the killing activity of CIK cells were compared. Results: the proliferation rate of CIK cells co-cultured with DC was higher than that of single CIK cells, but there was no significant difference compared with the proliferation rate of CIK cells co-cultured with DC loaded with antigen (P 0.05). The mature phenotype of DC cells in Ag-DC-CIK group was higher than that in DC group and Ag-DC group (P 0.01), and the maturation phenotype of CIK cells in Ag-DC-CIK group was higher than that in CIK group and DC-CIK group (P 0.01). The killing rate of SKOV3 in SKOV3-DC-CIK group was higher than that in CIK group, DC-CIK group and COC1-DC-CIK group (P 0.01). Conclusion: (1) Co-culture of DC and CIK loaded with SKOV3 freeze-thawed Ag can promote the maturation of DC,CIK cells, but DC loaded with SKOV3 freeze-thawed Ag can not significantly increase the proliferation rate of CIK cells compared with DC. (2) DC loaded with SKOV3 freeze-thawed Ag enhanced the specific killing effect of CIK cells on SKOV3.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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