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目的:探讨食管癌组织匀浆上清液体外模拟肿瘤微环境对人树突状细胞(DC)分化发育的影响,揭示肿瘤免疫逃逸机制,为DC疫苗的应用提供理论基础。方法:制备新鲜食管癌及癌旁组织匀浆上清液,ELISA检测其VEGF-A含量。密度梯度离心法分离人外周血单个核细胞,含rhGM-CSF和rhIL-4RPMI1640培养液诱导DC,第2天在继续诱导DC基础上设食管癌匀浆上清组、癌旁匀浆上清组、VEGF-A组,均隔天半量换液,第4天加入食管癌细胞株EC9706抗原,第6天加入脂多糖,第8天收集各组细胞。观察DC形态,流式细胞术(FCM)检测免疫表型,RT-PCR检测CD1a表达,CCK-8检测T细胞增殖率及杀伤率。结果:食管癌组织匀浆上清VEGF-A含量[(0.987±0.319)μg/L]明显高于癌旁[(0.152±0.105)μg/L,P<0.05];食管癌匀浆上清组细胞形态明显受到抑制,CD86分子阳性率(%)与正常DC相比由69±8降为42±11、CD1a由56±12降为27±12、CD11c由21±13降为18±13(P<0.01),CD1a基因几乎无表达,刺激T细胞增殖率(%)由112.5±7.2降为70.2±3.5(P<0.01),杀伤率(%)由62.4±0.6降为46.8±1.6(P<0.01);癌旁匀浆上清液组、VEGF-A组结果与正常DC组无统计学意义。结论:食管癌组织匀浆上清液所模拟的微环境对DC的诱导分化及其功能有明显的抑制作用,在该抑制作用中VEGF-A并不起主要作用。
Objective: To investigate the effect of esophageal cancer tissue homogenate supernatant on the differentiation of human dendritic cells (DCs) in vitro and to reveal the mechanism of tumor immune escape, providing a theoretical basis for the application of DC vaccine. Methods: Fresh esophageal cancer and para-cancerous tissue homogenate supernatants were prepared and the content of VEGF-A was detected by ELISA. Human peripheral blood mononuclear cells were isolated by density gradient centrifugation, and rhGM-CSF and rhIL-4RPMI1640 medium were used to induce DC. On the second day, DCs were further divided into three groups: esophageal homogenate supernatant group, paracancerous homogenate supernatant group , VEGF-A group, every half an hour to replace the liquid, adding EC9706 antigen on day 4, adding lipopolysaccharide on the 6th day and collecting the cells on the 8th day. The morphology of DCs was observed. The immunophenotype was detected by flow cytometry (FCM). The expression of CD1a was detected by RT-PCR. The proliferation rate and killing rate of T cells were detected by CCK-8. Results: The content of VEGF-A in the supernatant of esophageal cancer tissue was significantly higher than that in the adjacent [(0.987 ± 0.319) μg / L [(0.152 ± 0.105) μg / L, P <0.05] Cell morphology was significantly inhibited, the positive rate of CD86 molecules (%) compared with normal DC decreased from 69 ± 8 to 42 ± 11, CD1a decreased from 56 ± 12 to 27 ± 12, CD11c decreased from 21 ± 13 to 18 ± 13 ( (P <0.01). CD1a gene was almost not expressed, the proliferation rate of T cells was decreased from 112.5 ± 7.2 to 70.2 ± 3.5 (P <0.01) and the killing rate (%) was reduced from 62.4 ± 0.6 to 46.8 ± 1.6 <0.01). There was no significant difference between VEGF-A group and normal para-cancerous supernatant group and normal DC group. Conclusion: The microenvironment simulated by supernatants of esophageal cancer tissue can obviously inhibit the differentiation and function of DC, and VEGF-A does not play a major role in this inhibition.