亚硒酸钠对树突状细胞体外增强CTL杀伤K562细胞作用的研究  

Study of the Effects of Specific CTL Induced by DCs Treated with Sodium Selenite and ACL on Leukemia Cell line-K562

作　　者：杨磊[1] 刘复强[1] 王景文[1] 吴轶萍[1] 丁璟[1] 

机构地区：[1]首都医科大学附属北京同仁医院血液科

出　　处：《首都医科大学学报》2008年第2期137-141,共5页Journal of Capital Medical University

摘　　要：目的探讨经亚硒酸钠(Na2SeO3,Se)处理、K562细胞裂解物(又称为抗原细胞裂解物:ACL)冲击致敏的外周血单个核细胞(PBMNC)衍生的树突状细胞(DCs)体外诱导细胞毒性T淋巴细胞(CTL)杀伤白血病细胞的能力。方法1)DCs培养:用含3种细胞因子重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)和肿瘤坏死因子(TNF-α)的RPMI-1640(10%FBS)培养体系,培养健康人的PBMNC 4d,收获贴壁细胞;2)DCs分4组:Ⅰ组:单独DC培养组;Ⅱ组:加入0.5μmol/LSe的DC组;Ⅲ组:加入ACL的DC组;Ⅳ组:同时加入0.5μmol/LSe和ACL的DC组;3)效应细胞-CTL的诱导培养:用含细胞因子IL-2的1640(10%FBS)培养体系,将非贴壁细胞(淋巴细胞)作为效应细胞,单独或与各组DC共同孵育5d;4)CTL活性测定:用乳酸脱氢酶(LDH)释放试验,靶细胞为K562细胞。结果效应细胞与K562细胞按不同效靶比混合培养时,DC-Ⅳ组、Ⅲ组及Ⅱ组刺激后的T细胞比DC-Ⅰ组刺激后的T细胞及单纯T细胞对K562细胞的杀伤作用更显著,杀伤率随效靶比的增加而增加。其中E∶T=25∶1时,T细胞组及T-DC-Ⅰ、T-DC-Ⅱ、T-DC-Ⅲ、T-DC-Ⅳ组对K562细胞的杀伤率分别为:5.9%±2.4%、15.3%±2.3%、26.3%±3.7%、28.2%±4.5%、36.2%±3.7%。T-DC-Ⅳ组杀伤活性高于其他各组(P值均<0.01)。结论用含GM-CSF、IL-4和TNF-α的体外培养体系可从健康人PBMNC获得成熟的DCs,小剂量亚硒酸钠和K562细胞裂解液负载均可诱导出特异性杀伤靶细胞的CTL,且两者具有协同作用。Objective To investigate the effects of dendritic cells pulsed with lysed K562 cells frozen-thawed antigen（ACL） and treated with sodium selenite（Se） on inducing the eytotoxie T lymphocyte（CTL） to get specific anti-tumor activity in vitro. Methods 1） The culture of DCs： In RPMI-1640（ 10% FBS） medium containing 3 eytokines（ rhGM-CSF, rhIL-4 and TNF-α）, the peripheral blood mononuelear cells（PBMNC） from healthy donor were cultured for 4 days, the adherent cells （DCs） were harvested; 2） The 4 groups of DCs： DC-Ⅰ ： DCs only in the above culture system, DC-Ⅱ ： adding of 0.5 μmol/L Se, DC-Ⅲ： adding ACL, DC-Ⅳ： adding both 0.5 μmol/L Se and ACL; 3 ） Induction of the effective cell-CTL： non-adherent cells （suspending cells） cultured in the RPMI-1640 （10% FBS） with IL-2 for 2 days were cultured alone or with several DC groups for another 5 days; 4） Detection of CTL activity： The eytotoxieity was determined with LDH release method, using K562 cells as target cells. Results The CTLs induced in DC-Ⅱ , DC-Ⅲ and DC-Ⅳ groups had more potential effects on killing target cells than that induced in the DC-Ⅰ group, as well as in the T cell alone group, and their abilities increased along with elevation levels of the ratio of effective cells （E） to target cells （T）. At the ratio of 25： 1, the rates of K562 cells killing in the groups of T cell alone, T-DC-Ⅰ, T-DC-Ⅱ , T-DC-Ⅲ. and T-DC-Ⅳ were 5.9%±2.4% , 15.3% ±2.3% , 26. 3%±3.7%, 28.2% ± 4. 5% and 36.2% ± 3.7% respectively, the activity of CTL in T-DC-Ⅳ group was the strongest, it was significantly more potent as compared with those in other groups. Conclusion Dendritic cells can be successfully generated from PBMNC by culture system supplied with rhGM-CSF, rhIL-4 and TNF-α in vitro. Both DC-groups loaded with ACL loaded and treated with Se can induce specific CTL responses and they have a synergistic effects on killing leukemia cells.

关 键 词：白血病 树突状细胞 亚硒酸钠 冻融抗原 

分 类 号：R457.2[医药卫生—治疗学]