钩吻素子对免疫磁珠分离纯化的小鼠CD4^+T细胞体外增殖的影响  

Effect of koumine on proliferation of murine CD4^+ T cells purified by magnetic-activated cell sorting in vitro

作　　者：王志睿[1] 黄昌全[1] 张忠义[1] 张兰兰[1] 林敬明[1] 

机构地区：[1]南方医科大学珠江医院药剂科,广东广州510282

出　　处：《第一军医大学学报》2005年第5期562-564,共3页Journal of First Military Medical University

基　　金：广东省科技攻关项目(C30801)~~

摘　　要：目的探讨小鼠脾脏CD4+T细胞的分离方法,观察钩吻素子(Kou)对小鼠脾脏CD4+T细胞体外增殖的影响。方法以免疫磁珠分选法从小鼠脾脏细胞中分离CD4+T细胞,流式细胞术检测所得细胞的纯度,台盼蓝法检测细胞活力。将10~320μg/mlKou分别作用于经刀豆蛋白A或植物血凝素刺激的小鼠T淋巴细胞,四唑盐比色法检测细胞增殖情况,用酶联免疫法检测细胞上清中IL-2的含量。结果经过流式细胞仪测定分离后的小鼠脾脏CD4+T细胞纯度达到90.3%±5.8%;0.2%台盼兰染色细胞活力为94.9%±3.6%;增殖试验表明分离后未加Kou的CD4+T细胞对刀豆蛋白A、植物血凝素的刺激保持了良好的增殖能力,当浓度范围为20~320μg/ml时,Kou均能抑制淋巴细胞的增殖(P<0.05),且表现出一定的剂量相关性;不同浓度的Kou(20、100、200μg/ml)作用后,CD4+T细胞培养上清中的IL-2水平明显低于对照组(P<0.05)。结论免疫磁珠阴性分选法操作简单、快速,可获得较高纯化率、有活性的CD4+T细胞;Kou明显抑制小鼠CD4+T淋巴细胞增殖反应,此抑制作用可能与Kou抑制小鼠CD4+T细胞IL-2的分泌及免疫抑制作用相关。Objective To assess the separation efficiency of magnetic-activated cell sorting in the purification of CD4+ T cells from murine spleen, and observe the effects of koumine on the proliferation of the separated cells. Methods CD4+ T cells were isolated from murine spleen by magnetic-activated cell sorting (MiniMACS). Fluorescence-activated cell sortering was employed to determine the purity of CD4+ T cells before and after the separation procedure followed by evaluation of the cell viability using trypan blue staining. Concanavalin A- (ConA, 5 μg/ml) or phytahematoagglutinin (PHA,1 mg/ml)-induced murine T cells were treated with different concentrations of koumine (10-320 μg/ml), and their proliferation was determined by MTT colorimetry, and enzyme-linked immunosorbent assay was used to measure IL-2 level in the cell culture supernatant. Results The purity of CD4+ T cells reached (90.3±5.8)% after the purification with a cell viability of (94.9±3.6)%. Koumine (20-320 μg/ml) dose-dependently inhibited ConA- or PHA-induced proliferation of murine lymphocytes as compared with the controls (P<0.05). Koumine (20, 100, and 200 μg/ml) significantly decreased the level of IL-2 in comparison with the control group (P<0.05). Conclusions CD4+ T cells of high purity can be obtained from murine spleen using MiniMACS without impairing the viability of the cells. Koumine significantly inhibits the proliferation of murine CD4+ T cells due to its immunosuppressive effect and inhibition of IL-2 secretion.

关 键 词：CD4^+T细胞 免疫磁珠分选 钩吻素子 

分 类 号：R285.5[医药卫生—中药学]