不同冻存时间对 CIK 细胞免疫表型及杀伤活性的影响

摘要

Objective:To explore the effect of different cryopreservation time on CIK cells' immunophenotype and killing activities. Methods:PBMCs were obtained from 6 healthy adults,IFN - γ was added on day 0,anti - human CD3 and IL - 2 were added after 24 hours,adding fresh culture medium containing CD3 monoclonal antibody and IL- 2 were added according to the cell growth situation every 3 days,then continue culturing 15 days before cryopreser-vation. The cryopreserved CIK cells were reanimated monthly(1 ~ 5 months),routine cultured CIK cells as control group. The proportion of CD3 + CD4 + ,CD3 + CD8 + ,CD3 + CD56 + cells were analyzed by flow cytometry. The cytotox-icity of CIK cells were tested by CCK8 experiment. Results:There was no significant difference in the proportion of CD3 + CD4 + ,CD3 + CD8 + ,CD3 + CD56 + cells between fresh CIK and cryopreserved CIK,however,the proportion of CD3 + CD8 + ,CD3 + CD56 + cells in cryopreserved CIK reanimated after 4 months,5 months were lower than con-trol group[(70. 62 ± 6. 35)% ,(14. 36 ± 4. 28)% ,(67. 87 ± 5. 63)% ,(12. 61 ± 6. 21)% vs(84. 23 ± 5. 20)% , (22. 75 ± 3. 46)% ],P < 0. 05. The killing activity of cryopreserved CIK cells which reanimated after 4 months and 5 months at each effector - target ratio were weaker than control group(P < 0. 05). Conclusion:The immunophenotype and killing activity of CIK cells were influenced by cryopreservation time,cryopreserve more than 3 months showed lower activity.%目的：探讨不同冻存时间对外周血来源的 CIK 细胞免疫表型及其杀伤活性的影响。方法：采集6名健康患者外周血，分离外周血单个核细胞培养，第0h 加入 IFN -γ，24h 后加入 IL -2、抗 CD3单抗，每隔3天补充含 CD3单抗、IL -2的新鲜培养液，培养15天进行冻存。复苏冻存1、2、3、4、5月的 CIK 细胞为实验组，以常规培养至15天的 CIK 细胞为对照组。利用流式细胞仪检测各组 CIK 细胞中 CD3+ CD4+、CD3+ CD8+、CD3+ CD56+细胞的百分比，CCK8法检测各组 CIK 细胞对 K562细胞的杀伤活性。结果：冻存1、2、3月的 CIK细胞与对照相比 CD3+ CD4+、CD3+ CD8+、CD3+ CD56+细胞比例无明显差异（P >0.05），冻存4、5月的 CIK细胞中 CD3+ CD8+、CD3+ CD56+细胞比例较对照组明显下降[（70.62±6.35）%、（14.36±4.28）%、（67.87±5.63）%、（12.61±6.21）% vs（84.23±5.20）%、（22.75±3.46）%]，P <0.05。对靶细胞的杀伤能力在1、2、3月时与对照相比未见明显减弱（P >0.05），冻存4月、5月后的 CIK 细胞在各效靶比对 K562细胞的杀伤活性与对照相比明显减弱（P <0.05）。结论：冻存时间的长短对 CIK 细胞的免疫表型及杀伤活性均有一定的影响，且随着冻存时间的延长，CIK 细胞的杀伤活性减弱。建议冻存 CIK 细胞的最佳时间为1、2和3月。