【摘要】目的研究杀伤细胞对人白血病多药耐药（MDR）细胞系的杀伤作用。方法(1) 采用体外药敏试验（MTT法）检测两株MDR细胞系，即K562/VCR和K562/HHT 细胞的耐药性。(2)用优选方案诱导效应细胞：50 ng/ml的抗CD₃单克隆抗体（CD₃McAb）和50 U/ml的IL-2诱导的CD₃AK细胞（CD₃McAb activated killer cell）；400 U/ml的IL-2诱导淋巴因子激活的杀伤细胞（lymphokine activated killer cell，LAK）。(3)以³H-TdR掺入法测定效应细胞的增殖活性。(4)以³H-TdR释放法测定效应细胞对K562细胞及其两个MDR细胞系的杀伤活性。结果(1) K562/VCR、K562/HHT经冻存-复苏后无药培养1～2个月仍具有高度耐药性。(2)成功地诱导出CD₃AK^+/-细胞及LAK细胞。(3)CD₃AK⁺细胞的增殖活性较LAK细胞强；CD₃AK⁺细胞对K562细胞的杀伤活性较LAK细胞强，且维持时间较LAK细胞长。(4)CD₃AK⁺细胞对K562/VCR细胞的杀伤活性强于对敏感细胞K562，其对K562/HHT细胞的杀伤活性与其对K562细胞的杀伤活性比较，差异无显著性。LAK细胞对K562/VCR和K562/HHT细胞的杀伤活性比较，均与对K562细胞的杀伤活性差异无显著性。且CD₃AK⁺细胞对K562/VCR细胞的杀伤活性强于LAK细胞。结论CD₃AK⁺细胞对白血病细胞具有很强的杀伤活性，可望作为较理想的效应细胞用于耐药白血病的过继免疫治疗。

The cytotoxicities of anti-CD₃ monoclonal antibody activated killer cells against human multidrug resistant leukemic cell lines

XU Weiqun, GUO Shufen.

Children′s Hospital, Zhejiang University, Hangzhou 310003

【Abstract】ObjectiveTo study the cytotoxicities of the killer cells to human multidrug resistant (MDR) leukemic cell lines.Methods(1) The sensitivities of two MDR cell lines （K562/VCR and K562/HHT） reacted to vincristine (VCR), high harringtonine (HHT) and daunorubicin (DNR) were examined by MTT assay. (2) For induction of effector cells, 50 ng/ml of CD₃ monoclonal antibody (CD₃ McAb) and 50 U/ml of IL-2 were selected as the optimal condition for the induction of CD₃McAb activated killer cells (CD₃AK cells); 400 U/ml of IL-2 was used for the generation of lymphokine activated killer cells (LAK cells). (3)Proliferations of the effector cells were determined by ³H-TdR uptake assay. (4) Cytotoxicities of the killer cells to K562 as well as the two MDR cell lines of K562 were determined by ³H-thymidine (³H-TdR) release assay.Results(1) The multi-drug resistance consistently existed in both cell lines of K562/VCR and K562/HHT thawed after freezing and cultured for 1～2 months with drug-free medium. (2) CD₃AK^+/- and LAK cells were successfully induced. (3) The proliferation of CD₃AK⁺ cells was much stronger than that of LAK cells. The cytolysis of K562 cells by CD₃AK⁺ cells was higher than that of CD₃AK^- and LAK cells. The duration of the cytolytic activity of CD₃AK⁺ cells was longer than that of the LAK cells. (4) The hilling activing of CD₃AK⁺ cells showed significantly higher to K562/VCR cells than to K562 cells, while there was no difference of cytotoxicity between K562 /HHT and K562 cells. No significant difference was found in LAK-mediated cell killing either to K562/VCR and K562 /HHT cells or to K562 cells. The cell-mediated killing of K562/VCR cells by CD₃AK⁺ cells was significantly higher than that by LAK cells.Conclusion CD₃AK⁺ cells might be used as effector cells in adoptive immunotherapy of drug-resistant leukemic patients.

【Key words】LeukemiaAntibodies, monoclonalKiller cellsDrug resistance, multiple

白血病是小儿时期发病率最高的恶性肿瘤，化学治疗是其目前主要的治疗手段。耐药性的产生是化疗失败的主要原因。文献报道淋巴因子激活的杀伤细胞（lymphokine activated killer cell, LAK）对多数白血病耐药细胞系具有较高甚至有高于对敏感细胞系的杀伤活性^［1］，故可以用细胞过继免疫疗法辅助克服白血病的耐药。又据曹建平等^［2］报道，抗CD₃单克隆抗体激活的杀伤细胞（CD₃McAb activated killer cell,CD₃AK）与LAK细胞相比，具有体外增殖活性强、体内外杀伤活性强、IL-2用量少、体内应用副作用小等优点。CD₃AK细胞在体外和体内实验中均对白血病细胞具有高效杀伤作用^［3］。鉴于以CD₃AK细胞作为效应细胞杀伤人白血病多药耐药（MDR）细胞的实验研究国内外尚未见报道，为此我们进行了本研究。

材料和方法

一、材料

1.细胞系：K562为本院血液恶性肿瘤研究室保存的细胞系；K562/VCR、K562/HHT为本室诱导建立的MDR细胞系^［4］。

2.主要试剂：抗CD₃单克隆抗体（CD₃McAb)：军事医学科学院生物制剂发展中心产品。人重组IL-2（rhIL-2/IL-2）:上海华新生物制品公司产品。噻唑蓝（MTT）：瑞士Fluka公司产品。³H标记的胸腺嘧啶核苷（³H-TdR）：中国原子能研究所产品。

二、方法

1. 细胞培养：(1) 将白血病细胞系常规培养于RPMI-1640完全培养液［含10%新生牛血清（FCS），100 U/ml青霉素和100 μg/ml链霉素的RPMI-1640培养液］中。MDR细胞系于RPMI-1640完全培养液中加100 ng/ml原诱导药物以维持其耐药性,于实验前无药培养2～4周^［4］。 (2) 将CD₃AK及LAK细胞培养于效应细胞培养液［含10%FCS，5×10^-5 mol/L 二巯基乙醇（2-ME），2 mmol/L的L-谷氨酰胺（L-glu），25 mmol/L的N-2-羟基乙酯呱嗪乙烷硫酸(HEPES)，100 U/ml的青霉素和100 μg/ml的链霉素的RPMI-1640培养基^［5］］中。培养条件为37℃、5%CO₂、全湿度。

2. 靶细胞耐药性的检测（MTT法）^［6］：分别检测长春新碱（VCR）、高三尖杉酯碱（HHT）和柔红霉素（DNR）对K562/VCR 和 K562/HHT的半数抑制浓度（IC₅₀）。

3.效应细胞的诱导^［5］：常规分离正常人外周血单个核细胞（PBMC）。PBMC以低浓度的CD₃McAb和低浓度的IL-2共同作用诱导CD₃AK细胞（以CD₃McAb和IL-2共同激活48～72小时后,以含CD₃McAb和IL-2的效应细胞培养液维持培养诱导出CD₃AK⁺细胞，以仅含IL-2的效应细胞培养液中维持培养诱导出CD₃AK^-细胞，统称为CD₃AK细胞）；PBMC仅以IL-2作用诱导出LAK细胞。均为优选诱导方案。

4. 效应细胞增殖活性的测定：³H-TdR掺入法^［7］。将效应细胞加入96孔板中培养，于培养终止前6小时加入25 μci/ml的³H-TdR 20 μl/孔，培养结束后收集细胞，烘干后测定每分钟计数(count per minute，CPM)值。

5. 效应细胞杀伤活性的测定：³H-TdR释放法^［8］。(1) 标记靶细胞：取前1天传代的靶细胞，调整细胞浓度为1×10⁶/ml。在0.8 ml靶细胞悬液中加入90.9 μci/ml的³H-TdR 100 μl，标记4小时，每半小时摇1次，标记后洗3次，调整细胞浓度为2×10⁵/ml。(2)效应细胞洗3次，调整细胞浓度为5×10⁶/ml 。(3)效靶结合：96孔板中先加入靶细胞50 μl/孔，后加效应细胞100 μl/孔，空白孔仅加100 μl 效应细胞培养液，效靶比为50：1。培养18小时，于培养终止前半小时加入含0.6 mg的胰蛋白酶、10 μg DNA酶的混合酶液50 μl/孔，振荡后继续培养30分钟收集细胞，烘干后测定CPM值。

6.统计学处理：两组均值比较用t检验，多组均值比较用两因素或单因素方差分析，在586微机上用SPSS统计软件包进行分析。

结果

一、靶细胞耐药性的检测(表1)

表1K562/VCR和K562/HHT细胞系对VCR、

HHT、DNR的耐