体外富集Treg细胞对CTL杀伤MNK45胃癌细胞活性的影响及机制研究  

The influence of enriched CD4^+CD25^+ T regulatory cells on cytotoxic T lymphocytes killing MNK45 cells in vitro and its possible mechanism

作　　者：曾冬竹[1] 郑峻松[2] 王自强[1] 吴淼[1] 余佩武[1] 

机构地区：[1]第三军医大学西南医院普通外科肿瘤生物治疗中心,重庆400038 [2]第三军医大学医学检验系临床检验学教研室,重庆400038

出　　处：《消化外科》2006年第3期197-200,共4页Journal of Digestive Surgery

基　　金：国家自然科学基金资助项目(No.30200264);国家自然科学基金重大项目(No.3993430-2)

摘　　要：目的体外分析CD4+CD25+Treg(regulatory T cells)细胞对CD8+CTL(cytotoxic Tlym-phocytes)细胞杀伤MNK45胃癌细胞株活性的影响及机制。方法通过T细胞纯化柱分选小鼠脾细胞悬液T淋巴细胞,以MACS单阳性和双阳性分选法分别富集CD4+CD25+Treg细胞和CD8+CTL;通过不同浓度CD8+CTL与MNK45胃癌细胞混合培养,筛选有效杀瘤体外培养体系。通过Trans Well分隔培养和混合培养的方式向CTL与MNK45胃癌细胞株组成的体外抗肿瘤培养体系中加入CD4+CD25+Treg细胞,以瑞氏染色显微计数的方法观察Treg细胞对CTL杀伤MNK45胃癌细胞活性的影响,结合ELISA法检测各体系穿孔素浓度,分析影响活性的细胞及免疫学机制。结果质量浓度为2.0×105/ml的CTL与1.0×105/ml MNK45胃癌细胞混合培养,CTL能对MNK45胃癌细胞形成明显的杀伤作用(P<0.01);在此培养体系中以混合培养的方式加入质量浓度为5.0×104/ml CD4+CD25+Treg细胞即可明显抑制培养体系中CTL对肿瘤细胞的杀伤作用;如以Trans Well分隔培养的方式将CD4+CD25+Treg细胞与抗肿瘤培养体系分隔培养,达到同样抑制效能(实验组死亡肿瘤细胞百分率与对照组比较,P<0.05)的CD4+CD25+Treg细胞浓度须≥2.0×105/ml。结论CD4+CD25+Treg细胞能够对CTL杀伤MNK45胃癌细胞株的活性形成明显抑制,在Treg细胞与CTL作用的相应细胞和免疫学机制中,细胞接触机制占主导地位。Objective To investigate the effect of enriched CD4^＋ CD25^＋ T regulatory cells on cytotoxicity of CTL against gastric cancer cell line MNK45 cells in vitro and explore the possible underlying mechanism. Methods After T cells enrichment, MACS separating system was used to sort CD8^＋ CTL and CD4^＋ CD25^＋ Treg from spleen T lymphocytes. The optimal concentration of CTL and the best culture condition were explored by measuring the cytotoxichy of CTL against MNK45 at different doses. Then CD4^＋ CD25^＋ Treg were added into the culture system or in a separate well in transwell system. The influence of Treg on cytotoxicity of CTL was investigated by counting inactive MNK45 cells. Moreover, the cellular and immunological mechanism which might be involved was analyzed. Results In the culture system, CTL showed significant effect against MNK45 （ P 〈0.01）. When 1.0 ml （5.0 × 10^4/ml） was added to this reaction system, the amount of killed MNK45 cells decrease obviously （ P 〈0.05）. And when Treg cells were cultured seperatly in the TransWell system, Treg cells must be increased 2.0 × 10^5/ml to achieve the same inhibitory effect. Conclusions CD4^＋ CD25^＋ T regulatory cells could exert inhibitory effect on CD8^＋ CTL killing function in vitro, the cell-to-cell contact mechanism plays important roles in mediating this effect.

关 键 词：胃肿瘤 调节性T细胞 细胞毒性 T淋巴细胞 MNK45胃癌细胞株 

分 类 号：R735.2[医药卫生—肿瘤] R73-362[医药卫生—临床医学]