卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

In vitro anti-tumor effect of cytotoxicity T lymphocytes activated by dendritic cells loaded with freeze-thaw ovarian carcinoma antigen

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34+细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。

Objective To investigate the anti-tumor effect of cytotoxicity T lymphocytes (CTL) activated by dendritic cells (DC) loaded with soluble freeze-thaw antigen of ovarian carcinoma cells. Methods After isolated from umbilical cord blood by a high-gradient magnetic cell sorting system (MACS), CD34+ cells were cultured to differentiate into DC induced by a cocktail cytokines. Soluble antigen generated from SKOV3 ovarian carcinoma cells by freeze-thaw method was loaded on DC at day 7 of DC culture. The expression of DC phenotypes such as CD1α, CD83, CD80, CD54 and HLA-DR were examined by flow cytometry. DC function was evaluated by their ability to induce proliferation of allogeneic T cells in mixed lymphocyte reaction (MLR) assay, and their production of IL-12 using an ELISA method. Furthermore, the cytotoxicity of CTL activated by dendritic cells loaded with ovarian carcinoma antigen was evaluated by MTT method. Results Freeze-thaw ovarian carcinoma antigen up-regulated the expression of various differentiation antigens of DC, such as CD1α (73.35%±2.94% vs 34.1%±~2.35% ), CD83 (73.9%±8.46% vs 54.68%±3.26%), CD80 (91.95%±2.48% vs 52.53%±3.18%), HLA-DR (70.05%±2.35% vs 48.7%±2.07%), and CD54 (88.9%±5.52% vs 71.45%±2.29%), significantly higher than that of DC without loading antigen. Antigen-loaded DC also remarkably stimulated the proliferation of allogenic T cells and the secretion of IL-12 compared with control (P<0.05). CTL activated by antigen-loaded DC had more evident cytotoxicity (77.35% vs 45.88%) against ovarian carcinoma cells in vitro (P=0.0001). Conclusion CTL activated by DC loaded with freeze-thaw ovarian carcinoma antigen has a higher proliferative ability and effective cytotoxicity against ovarian carcinoma cells in vitro.