K652重组表达IL-6基因对NK细胞表型和功能的影响  

Effect of genetic modified K562 cells on NK cell markers and function

作　　者：李登瑞[1] 杨永辉[1] 李辉[1] 郭素敏[1] 朱桂云[1] 李秀武[1] 耿书军[1] 赵荣娣[1] 任雪飞[1] 高莉[1,2] 辛欣[1,2] 

机构地区：[1]河北省胸科医院,河北省肺癌防治中心,石家庄050041 [2]河北师范大学生命科学学院,石家庄050024

出　　处：《中国免疫学杂志》2015年第8期1070-1073,共4页Chinese Journal of Immunology

基　　金：2013年河北省省级重大医学研究课题(No.ZD2013063);国家自然科学基金(No.31101638)

摘　　要：目的:为了研究表达IL-6的重组K562细胞对自然杀伤细胞(NK)细胞的扩增数量、表型和功能的影响。方法:根据人类IL-6 c DNA的5'端序列,设计PCR引物以K562细胞c DNA文库的DNA为模板进行扩增,表达,转染,在K562细胞上表达IL-6基因,构建重组的K562工程细胞作为刺激细胞,以人外周血单个核细胞(PBMC)为扩增培养对象,使NK细胞在体外培养条件下得到大量的扩增。流式细胞仪分析NK细胞表型。将NK细胞作用到K562细胞,对其杀伤性进行功能分析,51Cr释放实验检测NK细胞对K562细胞杀伤水平的影响。结果:成功构建了表达IL-6的重组K562细胞,和PBMC共同孵育后,培养体系经过21 d的刺激后,IL-6重组K562细胞诱导PBMC扩增,CD56+CD16+CD3-细胞数量比诱导前扩增了(760±18)倍,CD56+CD16+CD3-细胞的纯度从培养前占PBMC的6%±0.4%,扩增后第3组结果比未扩增的多了91%±2%。细胞毒实验表明,在NK效应细胞∶K562靶细胞为5∶1时,扩增的NK细胞的杀伤率达到了92%±2%。结论:本方法以重组K562为刺激细胞,能够实现NK细胞体外的大规模制备,建立了优化的NK细胞体外扩增方法,且扩增的细胞杀伤K562细胞的活性较好。对于NK的大量扩增和应用于临床具有重要的指导意义。Objective： To study the influence of different culture conditions on charcic and inhibition activity of nature killer cells（ NK）,whether to join the modified K562 cells with IL-6 cytokine. Methods： According to the 5 ＇ end of the human IL-6 c DNA sequence,PCR primers designed to amplificate,express and transfect K562 cells c DNA library as a template for DNA. Genetic modified K562 cells as stimulating cells were prepared by expressing IL-6. To extract peripheral blood mononuclear cells（ PBMC） from human peripheral blood. PBMC were explanted by genetic modified K562 stimulated. The expansion was initiated by CO-culture of PBMC and irradiate genetic modified K562 cell. The number of NK cell increased by directed induced generation of genetic modified K562 cell. Immunophenotypic analysis of NK cell surface markers was performed by flow cytometry（ FCM）. 51 Cr release assay was employed to measure the specific lysis skilling of NK cell target K562 cells. Results： We have constracted genetic modified K562 cells by genetic engineering. As stimulated cell added into the PBMC,an average of 760 ± 18 fold expansion of CD56＋CD16＋CD3-cells was observed after 3 weeks of co-culture system. The NK cells population could proliferated more 91% ± 2% after expansion comparing with 6% ±0. 4% in PBMC before expansion by FCM. The cytotoxical activity of NK cells which was induced by genetic modified K562 cell was the strongest than induced by IL-6 cytokine alone. The expanded NK cells lysed 92% ± 2% of K562 targets in a 5∶ 1 effector to target ratio. In this case,the NK cells induced by genetic modified K562 cells against tumor cells was more lethal. Conclusion： PBMC based in vitro expansion of natural killer cells was set up by genetic modified K562 cells. The cytotoxicity of NK cells was the strongest induced by genetic modified K562 cell treated. These results had important guiding significance for the the NK large number of amplification and used in clinical.

关 键 词：重组K562 IL-6 自然杀伤细胞 扩增 51Cr释放实验 

分 类 号：R392[医药卫生—免疫学]