Lu Jing, Liu Kang-dong, Zhao Ming-yao, Yang Hong-yan, Huang You-tian, Qin Zhen-zhu, Bai Rui-hua, Dong Zi-ming
Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Nov;24(11):1055-8.
To study the effect of microenvironment simulated by esophageal carcinoma homogenate supernatant on the differentiation and development of human dendritic cells (DCs) and to investigate the mechanisms of tumor immune escape for the clinical application of DC vaccines.
Fresh esophageal carcinoma and peri-cancer tissues were collected to prepare homogenate supernatant and the content of VEGF-A was detected by ELISA. The peripheral blood monouclear cells were isolated by density gradient centrifugation and cultured with RPMI1640 medium including rhGM-CSF and rhIL-4 to induce to DCs. Then the esophageal carcinoma homogenate supernatant, peri-carcinoma homogenate supernatant and VEGF-A were added on the second day and half of the medium was changed every other day. Antigen of esophageal carcinoma cell line EC9706 was added on day 4 and lipopolysaccharide (LPS) was added on day 6. DCs were collected on day 8 for further study. Checked the morphology of DCs by microscope, the immunophenotype by flow cytometry, the gene of CD1a by RT-PCR and the proliferation and killing rate of T cell by CCK-8.
The content of VEGF-A in the homogenate supernatant of esophageal carcinoma was significantly higher than that of the peri-carcinoma (0.987+/-0.319 microg/L, 0.152+/-0.105 microg/L, P<0.05). The cell morphology in esophageal carcinoma homogenate supernatant group was inhibited. Besides, compared with normal DCs, the positive expression rate of CD86 decreased from 69+/-8 to 42+/-11, CD1a decreased from 56+/-12 to 27+/-12 and CD11c decreased from 21+/-13 to 18+/-13 (P<0.01). CD1a gene almost showed no expression. The proliferation capacity of T cells decreased from 112.53+/-7.16 to 70.18+/-3.47 (P<0.01), and their killing capacity of T cells decreased from 62.42+/-0.57 to 46.81+/-1.62 (P<0.01). However, the cells had no difference among peri-carcinoma homogenate supernatant group, VEGF-A group and normal DC group.
The tumour microenvironment stimulated by the esophageal carcinoma homogenate supernatant obviously has inhibitory effect on the differentiation and function of DCs.VEGF-A may not be the key factor in the process.
研究食管癌匀浆上清液模拟的微环境对人树突状细胞（DCs）分化发育的影响，探讨肿瘤免疫逃逸机制，为DC疫苗的临床应用提供依据。
收集新鲜食管癌组织及癌旁组织制备匀浆上清液，采用ELISA法检测VEGF-A含量。采用密度梯度离心法分离外周血单个核细胞，用含rhGM-CSF和rhIL-4的RPMI1640培养基培养诱导DCs。第2天加入食管癌匀浆上清液、癌旁匀浆上清液及VEGF-A，隔天换液1/2。第4天加入食管癌细胞系EC9706抗原，第6天加入脂多糖（LPS）。第8天收集DCs进行相关检测。通过显微镜观察DCs形态，流式细胞术检测免疫表型，RT-PCR检测CD1a基因，CCK-8法检测T细胞增殖及杀伤率。
食管癌匀浆上清液中VEGF-A含量明显高于癌旁组织（0.987±0.319μg/L，0.152±0.105μg/L，P<0.05）。食管癌匀浆上清液组细胞形态受抑制。此外，与正常DCs相比，CD86阳性表达率从69±8降至42±11，CD1a从56±12降至27±12，CD11c从21±13降至18±13（P<0.01）。CD1a基因几乎不表达。T细胞增殖能力从112.53±7.16降至70.18±3.47（P<0.01），T细胞杀伤能力从62.42±0.57降至46.81±1.62（P<0.01）。而癌旁匀浆上清液组、VEGF-A组与正常DC组细胞各项指标差异无统计学意义。
食管癌匀浆上清液模拟的肿瘤微环境对DCs的分化及功能有明显抑制作用，VEGF-A可能不是该过程的关键因素。
