抗人胸腺免疫球蛋白联合干扰素γ和白介素2诱导培养细胞因子诱导的杀伤细胞的效果

摘要

Aim:To investigate the activity and function of the cytokine induced killer cells ( CIK ) cultured by anti-human thymocyte immunoglobulin ( ATG) combined with recombinant human interferon γ( IFN-γ) and recombinant human interleukin-2(IL-2).Methods:The peripheral blood(10 mL) was collected from 9 cancer patients.The peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll density gradient centrifugation , and then were cultured with anti CD3 monoclonal antibody or different concentration (50,250,500 μg/L) of ATG combined with IFN-γand IL-2.Cell prolifera-tion was assessed using flow cytometry from D6 to D10.At the 13th day,the phenotype(CD3,CD4,CD8,CD56), the ex-pressions of activated markers(CD28,CD27,CD69 and NKG2D) and inhibitory markers(PD1 and CD152), as well as the levels of IFN-γand Granzyme-B secretion were analyzed by flow cytometry .Results:Compared with anti-CD3 group, the proportions of CD3 +CD4 +cells and CD3 +CD8 +cells were lower in ATG groups (P<0.001), while the percentages of CD3 -CD56 +natural killer cells and CD8 +CD69 +cells were elevated significantly in ATG groups (P<0.05).Further-more, the levels of IFN-γand Granzyme B secretion of CD56 +cells in all ATG groups were higher than that of anti-CD3 group(P<0.05).Conclusion:The CIK induced by ATG instead of CD3 monoclonal antibody combined with IFN-γand IL-2 exhibit better immune-competence and enhanced cytotoxic activity .%目的：探讨抗人胸腺免疫球蛋白（ ATG）诱导培养的细胞因子诱导的杀伤细胞（ CIK）的活性和功能，为CIK培养体系优化提供依据。方法：采集9例肿瘤患者外周血10 mL，分离单个核细胞，用CD3单抗或ATG （50、250、500μg／L）联合干扰素γ、白介素2诱导培养。培养至第6～10天，采用流式细胞术检测细胞增殖状况，培养至第13天，采用流式细胞术检测 CIK 免疫表型（ CD3、CD4、CD8、CD56）、激活性表面标志（ CD28、CD27、CD69、NKG2D）、抑制性表面标志（PD1、CD152）及Granzyme-B、干扰素γ分泌量。结果：4种培养方式下，细胞增殖状况无统计学意义（P＞0．05）。与CD3单抗相比，ATG培养的CIK中CD3＋CD4＋和CD3＋CD8＋细胞比例较低（P＜0．001）， CD3－CD56＋与CD8＋CD69＋细胞比例较高，CD56＋细胞干扰素γ和Granzyme-B分泌水平较高。结论：ATG诱导的CIK免疫活性优于用CD3单抗经典方案培养的CIK，抗肿瘤能力较强。