重组人纤维连接蛋白对CIK细胞增殖和杀伤活性的影响

目的 研究并探讨重组人纤维连接蛋白(RetroNectin,RN)对细胞因子诱导的杀伤细胞(CIK)增殖、免疫特性及对人类K562、LOVO细胞杀伤活性的影响。方法 提取患者外周血单个核细胞,标本分为四组,对照组(CIK组):按常规方法诱导CIK增殖;C-CIK组:提前通过CD3 Ab包被诱导;R-CIK组:提前通过RetroNectin包被诱导;R+C-CIK组:提前通过RetroNectin及CD3 Ab包被诱导,记录各组免疫效应细胞不同时间段的增殖情况。流式细胞术检测各组细胞不同时间段的免疫表型变化;并用LDH法检测各组细胞对肿瘤细胞K562、LOVO杀伤活性。结果 R+C-CIK组细胞增殖倍数高于其他三组,差异有统计学意义(P＜0.05);R+C-CIK组及R-CIK组的CD3⁺CD56⁺双阳性细胞所占百分比较C-CIK及CIK组高,d12和d15时有统计学差异(P＜0.05);在效靶比20∶1和40∶1时,R+C-CIK及R-CIK组对K562的杀伤活性高于C-CIK及CIK组,杀伤活性有统计学差异(P＜0.05),R+C-CIK组、R-CIK组及C-CIK组细胞对结肠癌细胞系LOVO的杀伤活性高于CIK组,有统计学差异(P＜0.05)。结论 RetroNectin联合抗CD3MAb包被技术应用于CIK体外培养可以获得增殖率和活性更高的免疫活性细胞,是值得临床推荐的一种有效的培养方法。

Effects of RetroNectin on CIK cell proliferation and cytotoxicity

Objective To investigate the recombinant human fibronectin(RetroNectin,RN)on proliferation,immunological characteristics and cytotoxicity of cytokine-induced killer cells(CIK).Methods Peripheral blood mononuclear cells(PBMCs)were cultured in vitro by precoating with RetroNectin(R-CIK group),CD3Ab(C-CIK group),RetroNectin combined with CD3Ab(R+C-CIK group)and traditional method(CIK group)were to generate CIK.The changes of growth rate,characterization,cytotoxicity and apoptosis of CIK were determined by cell counting,LDH assay and flow cytometry respectively.Results R+C-CIK cell showed a higher proliferation rate than other three groups.The difference was statistically significant(P＜0.05);The percentage of CD3/CD56 double positive cells in R+C-CIK and R-CIK group had a higher proportion than those in C-CIK and CIK group at day12 and day15(P＜0.05);Cytotoxicity of CIK towards K562 cells in R+C-CIK group and R-CIK group were significantly higher than that in C-CIK and CIK group,when effector-to-target ratio was 20∶1 and 40∶1(P＜0.05).We also found that the cytotoxicity of CIK towards LOVO cells in R+C-CIK group and R-CIK group were higher than that in C-CIK group and CIK group(P＜0.05).Conclusion RetroNectin and Anti-CD3-MAb synergistically promote antitumor efficiency of CIKs by increasing proliferation and cytotoxicity.