胃癌细胞FoxP3的表达及其与外周血单个核细胞(PBMCs)的相互作用

摘要

目的 检测叉头样蛋白3(forkhead box protein 3,FoxP3)在胃癌细胞中的表达分布,并探讨其在外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)和胃癌细胞共培养过程中的作用.方法 应用免疫荧光技术检测FoxP3蛋白的表达定位；建立胃癌细胞和PBMCs的体外共培养体系,然后用CCK-8法检测共培养后胃癌细胞的生长抑制情况；通过Real-time PCR技术检测共培养后胃癌细胞和PBMCs中FoxP3 mRNA表达情况；并采用流式细胞技术和Western blot技术检测共培养后两种细胞中FoxP3蛋白表达情况.结果 FoxP3表达于胃癌细胞的胞质和胞核.胃癌细胞与PBMCs共培养时能明显抑制胃癌细胞的增殖.直接和间接共培养后MKN28胃癌细胞的FoxP3蛋白的平均荧光强度(mean fluorescence intensity,MFI)均增加(P=0.031;P=0.015)；与单培养相比,来源于胃癌患者和健康对照组的淋巴细胞与胃癌细胞共培养后其FoxP3 MFI均明显增加(P=0.016;P=0.034),且IL-2能促进间接培养条件下PBMCs的FoxP3表达(P=0.024),而对直接共培养影响不大.共培养后胃癌细胞的FoxP3 mRNA和蛋白表达均增加,直接共培养较间接共培养增加更明显(P=0.035).结论 胃癌细胞与PBMCs的相互作用主要依赖细胞间的直接接触方式,FoxP3的表达变化在这一过程中发挥了重要作用,这可能跟肿瘤免疫逃逸有关.%Objective To detect forkhead box protein 3 (FoxP3) protein location in gastric cancer cells (GCCs) and to explore its roles in the interaction between peripheral blood mononuclear cells (PBMCs) and GCCs. Methods Immunofluorescence technique was applied to detect FoxP3 protein location; When GCCs-PBMCs cocultured systems were established,CCK-8 assay was used to detect cell growth inhibition of GCCs. Real-time polymerase chain reaction (PCR) technique was used to determine the expression of FoxP3 mRNA and protein in GCCs and PBMCs after cocultures. Flow cytometry and Western blot techniques were used to detect FoxP3 protein in the two types of cells. Results FoxP3 protein located both in nucleus and in cytoplasm of GCCs. The interaction between GCCs and PBMCs inhibited GCCs growth. FoxP3 mean fluorescence intensity (MFI) was increased after direct or indirect cocultures (P = 0. 031; P = 0. 015). Compared with monoculture, FoxP3 MFI in PBMCs obtained from GC patients or healthy control were increased after cocultured with GCCs (P = 0.016; P = 0.034). Moreover, IL-2 could promote FoxP3 expression in PBMCs after indirect coculture (P= 0.024), while there was no significant difference afterrndirect coculture. FoxP3 mRNA and protein levels were increased in GCCs and PBMCs, and their levels were higher in the direct coculture than those in the indirect one (P= 0. 035). Conclusions The interaction between tumor cells and PBMCs mainly depends on a direct cell-cell contact manner. FoxP3 plays a key role in their cross-talk, which probably contributs to tumor immune escape.