孔娟,龙世棋,王念雪,刘尧翠,王代琴,赵星
KONG Juan,LONG Shiqi,WANG Nianxue,LIU Yaocui,WANG Daiqin,ZHAO Xing

• 1:贵州医科大学免疫学教研室
• 2:贵州医科大学生物与工程学院
• 3:贵州医科大学组织工程与干细胞实验中心

摘要(Abstract)：

目的:探讨细胞因子诱导的杀伤细胞(CIK)联合西妥昔单抗对KRAS突变(DLD-1)及野生型(Caco-2)结直肠癌细胞的杀伤效应。方法:采用Ficoll密度梯度离心法分离正常外周血的单个核细胞(PBMC),在体外经抗-CD3单抗及多种细胞因子联合诱导生成CIK细胞;ELISA实验检测CIK细胞培养前后上清中干扰素-γ(IFN-γ)及转化生长因子-β(TGF-β)的水平,流式细胞检测DLD-1及Caco-2细胞表面表皮生长因子受体(EGFR)及CIK细胞表面分子的表达,采用实时无标记细胞分析仪(RTCA)检查CIK细胞联合西妥昔单抗对DLD-1及Caco-2细胞的杀伤作用。结果:DLD-1及Caco-2肿瘤细胞表面均高表达EGFR,培养前后CIK细胞亚群可见培养后CD8~+T、CD3~+CD56~+NKT及CD16~+CD56~+NK细胞比例均较培养前显著增高;与培养第一天比较,培养至第14天,CIK细胞培养基上清中细胞因子IFN-γ水平显著增高(P<0.01),而同时TGF-β的水平显著降低(P<0.01);RTCA检测发现CIK细胞联合西妥昔单抗对DLD-1及Caco-2杀伤作用明显高于单一的CIK细胞组。结论:CIK联合西妥昔单抗对EGFR受体阳性的野生型结直肠癌细胞与突变型结直肠癌细胞均有杀伤效果。
Objective: In order to solve the existing problems for CIK cells and cetuximab in clinical application,this project explored the antitumor effect of CIK cells combined with cetuximab against KRAS-WT and KRAS-mutant colorectal tumor cells,we expect to provide a new strategy for colorectal tumor treatment. Methods: Peripheral blood mononuclear cells(PBMC) from healthy donors were isolated by Ficoll density gradient centrifugation. CIK cells were generated from PBMCs with anti-CD3 antibody and several recombinant cytokines in vitro; IFN-γ and TGF-β levels in supernatant were measured by ELISA; The expression of EGFR on tumor cells and the phenotype of CIK cells were analyzed by flow cytometry; RTCA was applied to analyze cytotoxicity of CIK cells combined with cetuximab against colorectal tumors cells. Results: EGFR were expressed on tumor cell lines; The percentage of CD8~+T,CD3~+CD56~+NKT and CD16~+CD56~+NK were significant increased after cultured for14 days in vitro. The level of IFN-γ increased and TGF-β decreased in supernatant after cultured for14 d. Compared with singe CIK cells,the cytotoxicity of CIK cells combined with cetuximab against DLD-1 and Caco-2 cells increased. Conclusion: Equal efficacy of cytotoxicity against KRAS-WT and KRAS-mutant colorectal tumors cells were observed when CIK cells combined with cetuximab,it will lay the foundation for the next research work.

关键词(KeyWords)： 结直肠癌;西妥昔单抗;细胞因子诱导的杀伤细胞;联合治疗
colorectal cancer;cetuximab;cytokine-induced killer cell;combination therapy

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81360346);; 贵州省应用基础研究计划重大专项资助(黔科合J重大字2003);; 贵州省科技厅社会发展攻关项目资助[黔科合SY(2012)3094号];贵州省科技厅贵阳医学院联合基金资助项目[黔科合LG字(2011)011号]

作者(Author): 孔娟,龙世棋,王念雪,刘尧翠,王代琴,赵星
KONG Juan,LONG Shiqi,WANG Nianxue,LIU Yaocui,WANG Daiqin,ZHAO Xing

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