特异诱导脐血CD8^+抗白血病细胞毒淋巴细胞的研究(英文)  

Ex Vivo Specific Induction of CD8 Positive Anti-leukemia Cytotoxic Lymphocytes from Umbilical Cord Blood

作　　者：谭获[1] 刘芯[1] 叶絮[2] 

机构地区：[1]广州医学院第一附属医院肿瘤血液中心,广州510230 [2]广州医学院第二附属医院血液科,广州510260

出　　处：《中国实验血液学杂志》2007年第1期129-133,共5页Journal of Experimental Hematology

基　　金：广东省自然科学基金资助,No020721;广东省医学科研基金资助,NoA2003321;广州医学院科研基金资助,No.02-K-03~~

摘　　要：微小残留病灶(minimal residual disease,MRD)的存在是导致白血病复发的主要根源,特异性免疫治疗能有效地清除MRD,其中的策略之一就是诱导并回输白血病特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)。本实验的目的是研究脐血能否在体外诱导生成CD8+CTL,所生成的CD8+CTL能否特异性杀伤白血病细胞,从而确定脐血淋巴细胞的利用价值及用于特异性免疫治疗的可行性。通过联合细胞因子体外诱导脐血单个核细胞(mononuclear cells,MNC)分化为树突状细胞(dendritic cells,DC),同时负载U937冻融抗原;成熟DC刺激同源的脐血T淋巴细胞成为CTL,经MACS磁珠分选出CD8+CTL。用倒置显微镜、扫描电镜及流式细胞仪等方法检测DC,四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法测定杀伤活性。结果显示,10份人脐血标本均可培养出形态典型、功能成熟的DC。3组效应细胞CD8+CTL、CD8-CTL和T淋巴细胞(TL)组在相同效靶比(40∶1)对U937细胞株的杀伤率分别为(66.36±12.43)%、(34.47±8.19)%和(15.79±4.64)%,以CD8+CTL组最高;效靶比为40∶1时,CD8+CTL对U937细胞株的杀伤率(66.36%)高于对K562细胞株的杀伤率(41.97%)(P<0.05);而CD8-CTL对U937细胞株和K562细胞株的杀伤率无显著差异(P>0.05)。结论:用负载U937白血病细胞株抗原的成熟脐血DC可使脐血淋巴细胞诱导产生特异性的CD8+CTL;CD8+CTL对U937白血病细胞株的杀伤活性强于CD8-CTL的作用;CD8+CTL对U937白血病细胞株杀伤具有特异性。Minimal residual disease （MRD） is the principal root of relapsed leukemia. Application of specific immunotherapy is effective in eradication of MRD and one of the immunotherapeutic strategies is induction and re-transfusion of leukemia-specific cytotoxic T lymphocytes （CTLs）. This study was aimed to investigate the possibility of ex vivo induction and generation of CD8 positive CTLs of umbilical cord blood cell origin and to explore their potential of specific anti-leukemia cytotoxicity so as to evaluate the feasibility of application of cord blood cell derived lymphocytes to specific immunotherapy. Dendritic cells （DCs） were induced and generated ex vivo from cord blood mononuclear cells （MNC） with a cytokine cocktail, and loaded with frozen and thawed U937 leukemia antigen. The matured DCs were used to stimulate T lymphocytes derived from the same cord blood cell sample into CTLs. CD8 positive CTLs were then isolated by magnetic activated cell sorting （MACS）. Inverted microscopy, scanning electronic microscopy and flow cytometry were used to detect DCs and methyl thiazolyl tetrazolium （MTT） cytotoxicity method was used to assay the killing activity. The results showed that DCs with typical morphology and mature function were cultured from 10 human cord blood cell samples. The cytotoxicities of CD8 positive CTLs, CD8 negative CTLs and T lymphocytes （TLs） to U937 cells were （66.36 ± 12.43 ） %, （ 34.47 ± 8.19 ） % and （ 15.79 ± 4.64 ） % respectively under the same effector target ratio （40：1 ）. Among them, the anti-leukemia cytotoxicity of the CD8 positive group was highest. At effctor target ratio of 40 to 1, the cytotoxicity of CD8 positive CTLs to U937 cells （66.36%）was higher than that to K562 cells （41.97%） （ P 〈 0.05 ）, whereas the cytotoxicity of CD8 negative CTLs to U937 cells was not significantly different from that to K562 cells （ P 〉 0.05 ）. It is concluded that specific CD8 positive CTLs can be generated from cord blood lymphocytes by

关 键 词：CD8^+细胞毒性T淋巴细胞 免疫治疗 白血病 脐血 树突状细胞 

分 类 号：R730.51[医药卫生—肿瘤] R733.7[医药卫生—临床医学]