【摘要】： 目的：探討負(fù)載人卵巢癌細(xì)胞株SKOV3凍融抗原(Ag)的樹突狀細(xì)胞(DC)與細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(CIK)共培養(yǎng)后,對DC及CIK細(xì)胞增殖的影響；探討與負(fù)載人卵巢癌細(xì)胞株SKOV3或COC1凍融Ag的DC共培養(yǎng)的CIK細(xì)胞對SKOV3殺傷作用的影響。 方法：選擇12例上皮性卵巢癌患者,分離其外周血,獲得單個核細(xì)胞(PBMC),用相應(yīng)的誘導(dǎo)因子體外誘導(dǎo)出DC與CIK細(xì)胞。 分別收集處于對數(shù)生長期的SKOV3及COC1細(xì)胞,用細(xì)胞凍融法提取Ag,并于DC培養(yǎng)的第5天將Ag加入DC中培養(yǎng),使之成為負(fù)載Ag的DC。將經(jīng)Ag負(fù)載的DC與未經(jīng)Ag負(fù)載的DC分別和CIK細(xì)胞共培養(yǎng)后分組：實(shí)驗(yàn)組：將經(jīng)Ag負(fù)載的DC與CIK細(xì)胞共培養(yǎng)作為實(shí)驗(yàn)組(SKOV3Ag-DC+CIK組、COC1 Ag-DC+CIK組)。對照組：(1)將未經(jīng)Ag負(fù)載的DC與CIK共培養(yǎng)作為對照組1(DC+CIK組)；(2)CIK細(xì)胞單獨(dú)培養(yǎng)作為對照組2(CIK組)；(3)DC單獨(dú)培養(yǎng)作為對照組3(DC組)；(4)負(fù)載抗原的DC為對照組4(Ag-DC組)。 自培養(yǎng)第1天到第20天,用臺盼蘭拒染法動態(tài)監(jiān)測CIK細(xì)胞的增殖情況,觀察DC及Ag負(fù)載的DC對CIK細(xì)胞增殖的影響。 用流式細(xì)胞技術(shù)分析DC組、SKOV3Ag-DC組、SKOV3Ag-DC-CIK組中DC細(xì)胞的表型,觀察負(fù)載Ag及CIK對其增殖的影響。分析CIK組、DC-CIK組、SKOV3Ag-DC-CIK組中CIK細(xì)胞表型,觀察DC及Ag-DC對CIK細(xì)胞增殖的影響。 用乳酸脫氫酶釋放法檢測CIK組、DC-CIK組、Ag-DC-CIK組(包括SKOV3-DC-CIK及COC1 -DC-CIK組)對SKOV3的殺傷活性,對比觀察DC、SKOV3Ag-DC及COC1Ag -DC對CIK細(xì)胞殺傷活性的影響。 結(jié)果：與DC共培養(yǎng)后的CIK細(xì)胞的增殖速率大于單CIK細(xì)胞,但與同負(fù)載抗原的DC共培養(yǎng)的CIK細(xì)胞增殖率相比,差異無統(tǒng)計學(xué)意義(P0.05)；Ag-DC-CIK組中DC細(xì)胞成熟表型高于DC組及Ag-DC組(P0.01)；Ag-DC-CIK組中CIK細(xì)胞成熟表型高于CIK組及DC-CIK組(P0.01)；SKOV3-DC-CIK組對SKOV3殺傷率高于CIK組、DC-CIK組及COC1-DC-CIK組(P0.01)。 結(jié)論：(1)DC及負(fù)載SKOV3凍融Ag的DC與CIK共培養(yǎng)可促進(jìn)DC、CIK細(xì)胞的成熟,但負(fù)載SKOV3凍融Ag的DC與DC相比,不能明顯提高CIK細(xì)胞的增殖率； (2)負(fù)載SKOV3凍融Ag的DC可增強(qiáng)CIK細(xì)胞對SKOV3的特異性殺傷作用。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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