CAJ | 학술논문

目的探讨结肠癌组织匀浆上清液模拟肿瘤微环境对人树突状细胞(DC)分化发育的影响,以及血管内皮生长因子A(VEGF-A)在其中所起的作用.方法制备新鲜结肠癌及癌旁组织匀浆上清液.分离人外周血单个核细胞,含重组人粒细胞.巨噬细胞集落刺激因子(rhGM-CSF)和rhIL-4的1640培养液诱导DC,第2天在此基础上设结肠癌匀浆上清组、癌旁组织匀浆上清组、VEGF-A组及正常DC组,第4天加入结肠癌细胞株SW620抗原,第6天加入脂多糖,第8天收集各组细胞.ELISA检测肿瘤组织匀浆上清液中VEGF-A含量.观察DC形态,流式细胞术检测其免疫表型,RT-PCR检测CD1a表达,CCK-8检测T细胞增殖率及杀伤率.结果结肠癌组织匀浆上清VEGF-A含量明显高于癌旁组织(P＜0.05);与正常DC组相比,结肠癌匀浆上清组细胞形态明显受到抑制,数目减少,表面抗原表达率明显下降(P＜0.01),混合淋巴细胞反应能力及杀伤力也明显下降(P＜0.01);而VEGF-A组细胞数目及形态与正常DC组相比无明显改变,对所检测的Dc表面抗原并无明显抑制(P＞0.05),但在功能实验中它却起到了明显抑制T细胞增殖及杀伤功能的作用.结论结肠癌组织匀浆上清液所模拟的微环境对DC的诱导分化及功能有明显的抑制作用,在该过程中VEGF-A起到抑制T细胞免疫功能的作用,但该作用并非通过抑制DC共刺激分子表达而实现.
목적 탐토결장암조직균장상청액모의종류미배경대인수돌상세포(DC)분화발육적영향,이급혈관내피생장인자A(VEGF-A)재기중소기적작용.방법 제비신선결장암급암방조직균장상청액.분리인외주혈단개핵세포,함중조인립세포.거서세포집락자격인자(rhGM-CSF)화rhIL-4적1640배양액유도DC,제2천재차기출상설결장암균장상청조、암방조직균장상청조、VEGF-A조급정상DC조,제4천가입결장암세포주SW620항원,제6천가입지다당,제8천수집각조세포.ELISA검측종류조직균장상청액중VEGF-A함량.관찰DC형태,류식세포술검측기면역표형,RT-PCR검측CD1a표체,CCK-8검측T세포증식솔급살상솔.결과 결장암조직균장상청VEGF-A함량명현고우암방조직(P＜0.05);여정상DC조상비,결장암균장상청조세포형태명현수도억제,수목감소,표면항원표체솔명현하강(P＜0.01),혼합림파세포반응능력급살상력야명현하강(P＜0.01);이VEGF-A조세포수목급형태여정상DC조상비무명현개변,대소검측적Dc표면항원병무명현억제(P＞0.05),단재공능실험중타각기도료명현억제T세포증식급살상공능적작용.결론 결장암조직균장상청액소모의적미배경대DC적유도분화급공능유명현적억제작용,재해과정중VEGF-A기도억제T세포면역공능적작용,단해작용병비통과억제DC공자격분자표체이실현.
Objective To investigate the etfect of microenvironment simulated by colon carcinoma homogenate supernatant on the differentiation and development of human dendritic ceils (DCs), and to investigate the function of vascular endothelial growth factor A (VEGF-A) during this process . Methods Fresh colon carcinoma and peri-cancer tissues were collected to prepare homogenate supernatant. The pe-ripberal blood mononuclear cells were isolated and cultured with 1640 medium including rhGM-CSF and rhIL-4. Then the colon carcinoma homogenate supernatant, peri--carcinoma homogenate supernatant and VEGF-A were added to the cultures at day 2. Antigen of colon carcinoma cell line SW620 was added at day 4 and lipopolysaccharide (LPS) was added at day 6. DCs were collected at day 8 for further study. The con-tent of VEGF-A was tested by ELISA. The morphology and the immunopbenotype of DCs were checked by microscope and flow cytometry, respectively. The expression of CDIa was tested by RT-PCR, and the prolif-eration and killing rate of T cell was measured by CCK-8. Results The content of VEGF-A in the homoge-nate supernatant of colon carcinoma was significantly higher than that of the peri-carcinoma (P ＜ 0. 05). Compared with normal DCs, the cell morphology of colon carcinoma homogenate aupernatant group was in-hibited, and the cell number was decreased. Besides, the positive expression rate of DC surface markers de-creased (P ＜ 0.01). The capacity of mixed lymphocyte reaction (MLR) and killing capacity of T cells de-creased(P ＜0.01). However, there was almost no difference between VEGF-A group and normal DCs on the cell morphology and cell number, and VEGF-A had no obvious inhibition on the expression of DCs sur-face markers (P ＞ 0.05). But VEGF-A group had significantly inhibitory effect on the MLR and T cells kill-ing. Conclusion The tumor microenvironment simulated by the colon carcinoma homogenate supernatant obviously has inhibitory effect on the differentiation and function of DCs, and VEGF-A has the inhibitory effect on DC function, but the inhibitory effect is not through the inhibition of the expression of DC costimu-lators.