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

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10～20μg/ml Kou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.%±5.8%:0.2%台盼兰染色细胞活力为94.9%±.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20～20μg/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、200μgm1)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.

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

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.