体外培养不同时间后冻存对脐血来源CIK细胞生物学活性的影响  

Effect of cryopreservation on bioactivity of cytokine induced killer cells derived from human cord blood after in vitro culture of different time

作　　者：王小燕 王翔 赵令卉 郑榆坤 陈静娴 申重阳 邓红梅 张校通 漆巨兰 张蓉[2] 海泉 

机构地区：[1]成都清科生物科技(集团)有限公司,四川成都610000 [2]四川大学基础医学与法医学院,四川成都610041

出　　处：《中国肿瘤生物治疗杂志》2013年第3期342-347,共6页Chinese Journal of Cancer Biotherapy

摘　　要：目的:观察体外培养不同时间后冻存对人脐血来源细胞因子诱导杀伤(cytokine induced killer,CIK)细胞增殖、免疫表型、细胞毒活性以及细胞因子分泌的影响。方法:CIK细胞在体外培养6、9、12 d后冻存1个月,复苏后培养至72 h,其间每隔24 h检测细胞增殖情况,并采用流式细胞术检测复苏后细胞免疫表型、CCK-8法检测复苏后细胞对A549细胞的杀伤活性、ELISA方法检测复苏后CIK细胞分泌细胞因子IFN-γ和TNF-α的变化。结果:体外培养不同时间后冻存的CIK细胞在复苏后仍然显示出较好的增殖活性,其中,体外培养6 d后冻存组CIK细胞增殖活性明显高于体外培养9 d和12 d后冻存组CIK细胞,复苏72 h时,6、9、12 d冻存组CIK细胞数分别为(35.90±1.67)×106、(18.98±2.13)×106和(11.76±2.12)×106个(P<0.01)。体外培养6、9、12 d后冻存组CIK细胞复苏24 h后,各冻存组CD3+CD56+、CD3+CD8+细胞比例逐渐增加,且复苏72 h后6 d冻存组CIK细胞中CD3+CD56+和CD3+CD8+细胞比例最低。体外培养后冻存组CIK细胞在复苏24 h内对A549细胞的杀伤活性较低,但24 h后细胞毒活性有较大幅度的增加,且体外培养12 d后冻存组CIK细胞对A549细胞的杀伤活性高于6 d和9 d冻存组CIK细胞,如复苏后72 h,12、6、9 d冻存组CIK细胞对A549细胞的抑瘤率分别为(0.81±0.09)%、(0.59±0.06)%、(0.42±0.08)%(P<0.01)。ELISA检测结果显示,随着复苏时间的延长,体外培养不同时间后冻存的CIK细胞分泌IFN-γ和TNF-α的水平也逐渐上升;复苏72 h时,体外培养6 d后冻存组CIK细胞分泌IFN-γ的量要高于其他组,而体外培养12 d后冻存组TNF-α的分泌量则明显高于其他组。结论:体外培养不同时间后冻存对CIK细胞的生物学活性有一定影响,但复苏后经短时间培养后基本恢复,提示CIK细胞可以在培养至12 d后进行冻存。Objective：To observe the effect of in vitro culture of different time on proliferation,immunophenotype,cytotoxicity and cytokine secretion of human cord blood derived cytokine induced killer（CIK） cells after cryopreservation.Methods： CIK cells were cryopreserved for 1 month after in vitro culture for 6,9 and 12 d.The cell proliferation was detected every 24 h when they were recovered and cultured for 72 h.The immunophenotype,cytotoxicity on A549 cells and the secretion of IFN-γ and TNF-α of CIK cells after recovery were detected by flow cytometry,CCK-8 assay and ELISA assay,respectively.Results： CIK cells,in vitro culture of different time before cryopreservation still showed a superior proliferation activity after recovery,among which the proliferation of CIK cells in vitro cultured for 6 d before cryopreservation,was significantly higher than those in vitro cultured for 6 and 12 d before cryopreservation.The CIK cell counts were（[35.90±1.67]×106,[18.98±2.13]×106,and [11.76±2.12]×106,P0.01） in 6,9 and 12 d cryopreservation groups after recovery for 72 h.After recovery for 24 h,the proportions of CD3＋CD56＋ and CD3＋CD8＋ cells increased gradually in all the three 6,9 and 12 d cryopreservation groups.Moreover,both two cell proportions in 6 d cryopreservation group after recovery for 72 h was the lowest.The cytotoxicity of CIK cells on A549 cells within 24 h of recovery in all the three cryopreservation groups was much low.However,their cytotoxicity increased considerably after 24 h.The cytotoxicity of CIK cells on A549 cells in 12 d cryopreservation group was higher than those in the 6 and 9 d cryopreservation groups.For example,the anti-tumor rate of CIK cells on A549 cells in 6,9 and 12 d cryopreservation groups after recovery for 72 h was（[0.81±0.09]%,[0.59±0.06]% and [0.42±0.08]%,P0.01）.ELISA assay results showed that,the levels of IFN-γ and TNF-α secreted by CIK cells increased along the recovery time in all cryopreservation groups that had been in vitro cultured for vario

关 键 词：细胞因子诱导杀伤细胞 冻存 细胞毒活性 免疫表型 

分 类 号：R392.33[医药卫生—免疫学] R734.2[医药卫生—基础医学]