B-NHL患者NK细胞中CD16ζ表达及利妥昔单抗与LAK细胞的联合抗瘤作用  

Expression of CD16ζ in NK Cells of B-cell Non-Hodgkin's Lymphoma Patients and In Vitro Killing Effect of Rituximab Combined Lymphokine-activated Killer Cells on B-NHL Cells

作　　者：史艳侠[1] 张晓实[1] 夏建川[1] 李永强[1] 徐瑞华[1] 韩文杰[2] 张景航[2] 管忠震[1] 姜文奇[1] 

机构地区：[1]华南肿瘤学国家重点实验室 [2]新乡医学院基础医学系,河南新乡453003

出　　处：《癌症》2007年第8期837-842,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30500610);广东省自然科学基金(No.04009388);广东省医学科研基金(No.B2005055)~~

摘　　要：背景与目的:自然杀伤细胞(nature killer cell,NK)是抗体依赖细胞介导的细胞毒作用的主要效应细胞,肿瘤患者普遍存在NK细胞活化功能的缺陷可能会影响单克隆抗体的治疗效果。因此如能逆转NK细胞的CD16ζ链信号转导的功能缺陷,并与单克隆抗体联合免疫治疗,可能会产生协同抗肿瘤作用。本研究的目的是了解B细胞非霍奇金淋巴瘤(B-cellnon-HodgkinI's lymphoma,B-NHL)患者是否存在NK细胞的活化障碍,体外白细胞介素-2(interleukin-2,IL-2)是否能完全逆转其活化障碍,并观察利妥昔单抗与LAK细胞联合对肿瘤细胞的杀伤作用。方法:使用密度梯度离心方法分别分离69例B-NHL患者和30例健康志愿者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),将两种PBMC在体外与1000U/mlIL-12共同培养制备LAK细胞,流式细胞仪检测PBMC和LAK细胞中CD16ζ链的阳性率和平均荧光强度。流式细胞仪检测Raji细胞表面CD20的表达;AnnexinⅤ/PI方法检测利妥昔单抗单药对Raji细胞的促凋亡作用,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验进行杀伤活性的检测。结果:在B-NHL组和健康对照组,CD56+细胞表达CD16ζ链的阳性率为(63.3±16.4)%、(97.8±3.1)%(P<0.001),CD16ζ链MFI值分别为1.3±1.3和3.6±1.7(P<0.001)。在体外1000U/ml的IL-2共培养的LAK细胞中,两组CD16ζ链的阳性率分别为(99.3±4.1)%和(99.7±3.9)%,其MFI值分别为29.2±12.5和31.4±13.8,均无显著性差异(P=0.15和P=0.44)。40μg/ml利妥昔单抗可以完全结合细胞表面CD20抗原,在24h时才开始出现对Raji细胞的明显的凋亡作用。利妥昔单抗与LAK细胞联合对Raji细胞的杀伤率在不同的浓度组均明显高于不加利妥昔单抗组(P<0.05)。LAK细胞与Herceptin(40μg/ml)联合的杀伤率与不加Herceptin组相比,在各效靶比浓度梯度均无明显提高(P>0.05)。LAK细胞与利妥昔单抗联合对Jurket细胞的杀伤率在各效靶比浓度梯度均与不加利妥昔单抗�BACKGROUND ＆ OBJECTIVE： Natural killer （NK） cells are the main effector of antibody-dependent cellular cytotoxicity （ADCC）. The activation disorder of NK cells in cancer patients may affect the treatment effect of monoclonal antibody. Reversing the dysfunction of signal transduction of CD16ζ chain in NK cells and combining lymphokine-activated killer （LAK） cells with rituximab may give rise to synergistic effect. This study was to find out whether the activation disorder of NK cells exist in B-cell non-Hodgkin＇s lymphoma （B-NHL） patients, whether interleukin-2 （IL-2） can reverse the activation disorder in vitro, and whether the combination of rituximab and LAK cells can produce synergistic antitumor effect. METHODS： Peripheral blood mononuclear cells （PBMCs） were isolated from 69 B-NHL patients and 30 healthy donors by density gradient centrifugation method, and cultured with IL-2 （1 000 U/ml） to prepare LAK cells. The positive rate and median fluorescence intensity （MFI） of CD16ζ chain in PBMCs and LAK cells were detected by flow cytometry （FCM）. The expression of CD20 on Raji cells was also detected by FCM. The apoptosis of Raji cells after treatment of rituximab was detected by FCM with Annexin Ⅴ/PI staining. The cytotoxicity was assessed by lactate dehydrogenase （LDH） release experiment. RESULTS. The positive rate and MFI value of CD16ζ chain on CD56^＋ cells were significantly lower in B-NHL group than in control group [（63.3±16.4）% vs. （97.8±3.1）%, P〈0.001; 1.3±1.3 vs. 3.6±1.7, P〈0.001]. There was no significant difference in the positive rate and MFI value of CD16ζ on LAK cells between the 2 groups [（99.3±4.1）% vs. （99.7±3.9）%, P=0.145; 29.2±12.5 vs. 31.4±13.8, P=0.44]. At the concentration of 40 μg/ml, rituximab completely combined CD20 antigens on cell membrane. The obvious enhancive effect of rituximab on cell apoptosis appeared at 24 h after treatment. The killing rate of Raji cells was significantly higher in rituximab

关 键 词：利妥昔单抗 LAK细胞 免疫治疗 淋巴瘤 

分 类 号：R733.1[医药卫生—肿瘤]