重组人纤维连接蛋白对无血清培养的CIK细胞增殖及对K562细胞杀伤活性的影响

摘要

Objective To investigate the effect of RetroNectin on the proliferation, immunologic characteristics and cytotoxicity of cytokine-induced killer cells (CIK). Methods Peripheral blood mononuclear cells (PBMC) were collected from healthy donors and divided into two groups: RN group and Non-RN group. The proliferation of CIK cells was tested by cytometirc analysis. The cytotoxic activity of CIK cells was determined by CFSE/PI double-labeling assays. The cytokine secretion profile of RN group and Non-RN group was analyzed by BDTM-CBA Human Thl/Th2 kit. Results The RN-induced group had a higher proliferation rate than that of the Non-RN group (P<0.05). The two groups differed significantly in CD3 + CD56+ cells at the fifth day (P<0.05), and the significant difference was maintained till the 25th day; but there was no significant difference between the two groups in CD3 + CD8+ T cells (P>0.05). Cytotoxity and the secretion profile of IFN-y and TNF-a differed significantly between the two groups. As the culture time went on, the secretion amount of IFN-y and TNF-a was increased. There was no significant difference between the two groups in the amount of IL-4. Conclusion RetroNectin plus serum-free culture is a safe and effective way to culture CIK cells because they have faster proliferation rate and high cytotoxic activity.%目的 探讨重组人纤维连接蛋白(RetroNectin,RN)诱导的细胞因子诱导的杀伤细胞CIK增殖、免疫特性及对人类K562细胞杀伤活性的影响.方法 密度梯度分离法分离外周血单个核细胞后分成两组,应用CD3单抗、IFN-γ、IL- 2培养CIK细胞,一组含RN(RN组),一组不含RN(Non-RN组).记录两组细胞增殖速度；用流式细胞术动态测定免疫细胞表型；用CBA细胞因子试剂盒检测IFN-γ、TNF-α及IL-4的分泌；用CFSE/PI双标法测定CIK细胞对人红白血病细胞株(K562)的体外杀伤活性.结果 RN组细胞生长明显快于Non-RN组(P＜0.05)；CD3+CD8+的细胞随着时间的延长逐渐增多,但两组间比较无统计学差异；CD3+ CD56+细胞所占百分比在第5天和第10天时两组间比较均无统计学差异(P＞0.05),在第15天时RN组有大幅度升高,第15、20、25天两组比较均有统计学差异(P＜0.05).细胞毒杀伤活性结果显示,RN组与Non-RN组对K562细胞毒活性亦有统计学差异.两组间细胞因子IFN-γ、TNF-α随着培养时间的延长,分泌量逐渐升高.结论 使用RN诱导的CIK细胞增殖速度快,杀伤肿瘤细胞活性高,是一种安全高效的细胞培养方法.