重组人纤维蛋白片段对CIK细胞增殖的影响及其可能机制研究  

Effect of RetroNectin on the Proliferation of CIK Cells and the Possible Mechanisms

作　　者：韩颖[1] 于津浦[2] 李慧[2] 曹水[2] 任宝柱[2] 齐静[2] 安秀梅[2] 张廼宁 任秀宝[2] 郝希山[2] 

机构地区：[1]辽宁医学院 [2]天津市肿瘤防治重点实验室天津医科大学附属肿瘤医院生物治疗科,天津市300060

出　　处：《中国肿瘤临床》2010年第2期71-75,共5页Chinese Journal of Clinical Oncology

基　　金：国家十五科技攻关引导项目基金资助(编号:2005BA740C)~~

摘　　要：目的:探讨重组人纤维蛋白片段(RetroNectin)对CIK细胞的增殖作用及其可能机制。方法:提取患者外周血单个核细胞,Ⅰ组细胞培养于前一天用RetroNectin和CD3m Ab包被好的培养瓶中,Ⅱ组培养于普通培养瓶中。第2天起按照常规CIK细胞培养方法分别培养,第14天收获。细胞计数法测定CIK细胞增殖;LDH法测定细胞杀伤活性;流式细胞术分析细胞表型并检测细胞周期;Annexin V/PI法检测细胞凋亡:封闭CD49d^+及CD49e^+后细胞计数法测定CIK细胞增殖;流式细胞术分析细胞表型;Western blot检测Vav1蛋白表达水平。结果:RetroNectin对CIK细胞增殖有明显促进作用(P<0.05),经RetroNectin包被后可以增加CIK细胞中CC25^+T细胞比例(P<0.05),对于结肠癌细胞系HCT-8 RN-CIK较CIK显示出了更强的杀伤活性(P<0.05),而对于白血病细胞系K562两组细胞杀伤活性无显著性差异(P>0.05)。RetroNectin可以将CIK细胞阻滞于G_1期而促进增殖,同时抵抗高活化细胞的凋亡。RetroNectin对CIK细胞中的CD49d^+CD49e^+T细胞亚群有明显上调作用(P<0.05)。拮抗CD49d/CD49e后RetroNectin对CIK细胞增殖作用消失(P>0.05),对CD25^+T细胞的上调作用也消失。Western blot结果显示Vav1蛋白表达与CD25^+T细胞变化相一致。结论:重组人纤维蛋白片段(RetroNectin)能够通过调控细胞周期,抵抗凋亡而促进CIK细胞增殖,其机制可能为RetroNectin与CIK细胞上VLA-4及VLA-5两个位点结合进而通过Vav1蛋白作为第二信号活化T细胞。Objective： To investigate the effect of RetroNectin on CIKs cells and the related mechanisms. Methods： Peripheral blood mononuclear cells （PBMC） were collected from patients and divided into two groups： group Ⅰ and group Ⅱ. Samples in group Ⅰ were seeded into culture flask precoated with RetroNe tin and CD3mAb to induce CIKs. While samples in group Ⅱ were seeded into common culture flask. The proliferation of CIKs was detected by cytometric analysis. The cytotoxic activity of CIKs was determined by LDH assays. The phenotype changes and cell cycle of CIKs were identified by flow cytometry. The apoptosis of cells was detected by Annexin V/PI. Western blot was employed to detect the level of protein Vavl. The CD49d and CD49e were blocked by anti-CD49d and anti-CD49e and the proliferation of cells was tested by cytometric analysis after the blockage. The phenotype changes of cells were identified by flow cytometry after the blockage. Results： RetroNectin enhanced the proliferation of CIKs （P〈0.05）. Flow cytometric analysis showed that RetroNectin significantly increased the number of CD25^＋ T cells （P〈0.05）. RN-CIK was more active than CIK in killing HCT-8 cell lines in vitro （P〈0.05）. RetroNectin could block the CIKs at G1 phase （P〈0.05） and resist apoptosis. There was no significant difference in the proliferation between the two groups after the blockage with CD49d and CD49e （P〉0.05）. The expression of protein Vavl was associated with CD25^＋T cells. Conclusion： RetroNectin enhances the proliferation of CIKs by influencing the cell cycle, resisting apoptosis possibly through the site of CD49d and CD49e, and inducing T cell activation as the second signaling through Vav1.

关 键 词：重组人纤维蛋白片段 CIK细胞 Vav1 

分 类 号：R341[医药卫生—基础医学]