冻存对细胞因子诱导的杀伤细胞免疫表型及细胞内因子表达的影响

Effects of cryopreservation on CIK immunophenotype and intracellular cytokine expression

目的:探讨冻存对外周血来源的细胞因子诱导的杀伤细胞(CIK)免疫表型及细胞内因子表达的影响。方法:采集10例癌症患者的外周血,采用Ficoll两步法分离外周血单个核细胞,用细胞因子诱导培养得CIK。收集冻存3个月后复苏4、24、72 h的CIK,采用流式细胞术及胞内染色法检测细胞穿孔素、颗粒酶B及干扰素-γ的表达水平,采用AnnexinV/PI双染法检测细胞活性。以未冻存的新鲜CIK作对照。结果:冻存前及复苏4 h后CIK颗粒酶B及干扰素-γ表达水平差异无统计学意义(t=1.029、0.237,P>0.05)。冻存前及复苏4、24、72 h后CIK穿孔素表达水平分别为(35.97±7.12)%、(10.00±6.04)%、(17.60±3.92)%和(35.20±6.23)%,差异有统计学意义(F=47.480,P<0.001),冻存后CIK穿孔素表达水平显著降低,随着复苏时间的延长,穿孔素表达水平逐渐恢复正常。复苏4、24、72 h后CIK活细胞比例逐渐增多(P<0.05),复苏72 h后活细胞比例接近100%,细胞活性恢复正常。结论:冻存可能使CIK细胞亚型发生改变。

Aim： To explore the effects of cryopreservation on cytokine-induced killer cell （ CIK ） immunophenotype and intracellular cytokine expression .Methods：Peripheral blood mononuclear cells from ten patients with cancer were iso-lated by Ficoll-Plaque density gradients and CIKs were obtained by incubating with appropriate cytokines .The secretion levels of perforin, granzyme-B and interferon-γin CIK cryopreserved for 3 months and recovered for 4,24,and 72 h were detected by flow cytometry assay and intracellular staining , and cellular activity were detected by PI and AnnexinV stai-ning.The fresh CIKs were the control .Results：There was no significant difference in the levels of granzyme-B or interfer-on-γbetween fresh CIK and those recovered for 4 h（t=1.029,0.237,P＆gt;0.05）.However, the level of perforin in fresh CIK, those recovered for 4,24,and 72 h cells were （35.97 ＆#177;7.12）%,（10.00 ＆#177;6.04）%,（17.60 ＆#177;3.92）% and （35.20 ＆#177;6.23）%, and there was significant difference （F=47.480,P＆lt;0.001）;the expression level in perforin cryopre-served CIK decreased and gradually reached to the normal level with time .Cellular activity of CIK also recovered gradually （P＆lt;0.05）.Conclusion：Cryopreservation might change the cell subtypes of CIK .