抗人卵巢癌-抗人CD3单链双特异性抗体体外介导的细胞毒作用及其机制  

Cytotoxicity of peripheral blood lymphocyte-mediated by an anti-human ovarian carcinoma-anti- human CD3 bispecific single-chain antibody （ BHL-I ） in vitro and its mechanisms

作　　者：杨介钻[1] 李兰娟[1] 何剑琴[1] 王祥斌[2] 王小宁[3] 

机构地区：[1]浙江大学医学院附属第一医院传染科卫生部传染病重点实验室,杭州310003 [2]北京安波特基因工程技术有限公司 [3]华南理工大学生物科学与工程学院

出　　处：《中华肿瘤杂志》2008年第1期8-11,共4页Chinese Journal of Oncology

基　　金：国家重点基础研究发展规划（973）资助（2003CB515506、2001CB510008）

摘　　要：目的了解抗人卵巢癌-抗人CD3（BHL—I）单链双特异性抗体（scBsAb）体外介导的外周血淋巴细胞（PBL）对靶细胞SKOV3的细胞毒作用及可能的机制。方法二苯基溴化四氮唑蓝（MTr）法检测BHL-I体外介导PBL对SKOV3细胞的杀伤作用；逆转录-聚合酶链反应（RT—PCR）检测BHL—I介导的细胞毒作用过程中效应细胞PBL对穿孔素（perforin）、颗粒酶（GrB）mRNA表达的影响；酶联免疫吸附（ELISA）法检测细胞培养上清液中人肿瘤坏死因子-α（hTNF—α）和人干扰素-γ（hIFN-γ）含量的变化。结果BHL-I体外介导PBL对SKOV3细胞的细胞毒作用显著高于对MCF-7细胞的作用（P〈0.01），并且在效靶比为12．5：1、作用时间为36h、BHL-I浓度为25μg/ml时，PBL对靶细胞的细胞毒作用最为显著；BHL—I体外介导的细胞毒作用中，PBL表达perforin、GrBmRNA及混合细胞培养上清液中hTNF—α和hIFN-γ的含量均显著升高，并且白介素2（IL-2）的存在有利于这些因子的表达。结论BHL—I介导PBL对靶细胞的细胞毒作用具有一定的特异性，并且IL-2能增强BHL—I介导PBL的细胞毒作用。Objective To investigate the phenomenon that SKOV3 was killed by the PBL mediated by BHL-Iin vitro and the possible related mechanisms. Methods MTT colorimetric assay was used to analyze in vitro ff PBL is cytotoxicitic to the target cells at different ratios of activated PBL and target cells （25：1,12.5：1,6.25 ： 1,3.2： 1 ）, BHL-I mediating time （ 24,36,48 h ）, targets （ SKOV3, MCF-7 ） and concentration of BHL-I （100,50,25,12.5,6.25,3.2,1.6,0 μg/ml）. The levels of perforin, GrB mRNA and the concentrations of hIFN-γ and hTNF-α, which expressed and secreted by the effector （PBL） in the process of target cell being killed, were evaluated by RT-PCR or ELISA, respectively. Results The cytotoxicity of PBL to SKOV3 cells was more efficient than to MCF-7 cells （P 〈0.01 ）, and the PBL exhibited most effective cytotoxicity to SKOV3 cells when the concentration ratio of PBL to SKOV3 was 12.5：1, and the mediated time of 25 μg/ml BHL-I was 36 h. Furthermore, The PBL expressed a higher level of perforin, GrB mRNA and hTNF-α, hIFN-γ when the BHL-I was present, and the IL-2 increased the level of these cytokines expressed by PBL. Conclusion The anti-human ovarian carcinoma x anti-human CD3 bispecific single-chain antibody BI4L-I can induce PBL efficiently to kill the SKOV3 cells containing related antigens. The cytotoxic activity may be associated with the cytokines （perforin, GrB, hTNF-α, hIFN-γ） which expressed or secreted by activated PBL, while the IL-2 can enhance the cytotoxicity of PBL mediated by BHL-I.

关 键 词：抗人卵巢癌-抗人CD3 单链双特异性抗体 细胞毒作用 细胞因子 

分 类 号：R737.31[医药卫生—肿瘤]