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目的分析葫芦素B(CuB)对多克隆丝裂原激活的小鼠淋巴细胞活化、增殖、凋亡和γ-干扰素(IFN-γ)表达的影响,探讨其免疫调节作用的机制。方法经CuB处理的小鼠淋巴细胞,在刀豆蛋白(ConA,5μg/ml)或者佛波醇酯(PDB,10-7 mol/L)和离子霉素(Ion,0.5μg/ml)刺激后,细胞计数观察CuB对小鼠淋巴细胞增殖的抑制作用;流式细胞术分析CD69和IFN-γ的表达及亚二倍体峰和线粒体膜电位的变化;免疫印迹分析Caspase 3的活化水平。结果细胞计数分析显示,CuB对小鼠淋巴细胞增殖的抑制呈剂量依赖关系。对细胞DNA含量分析表明,随着CuB浓度的增加,处于亚二倍体峰(即凋亡峰)的细胞数量增加。同样,经CuB处理后,早期活化标志CD69和IFN-γ在CD3+T细胞中的表达均受剂量依赖性抑制。JC-1染色结果表明,10μmol/L CuB能显著使小鼠淋巴细胞的线粒体膜电位发生下降(P<0.01)。免疫印迹分析表明,10μmol/L CuB处理24 h后,Caspase 3明显活化。结论 CuB对小鼠淋巴细胞的增殖和活化具有明显抑制作,可以诱导体外培养的小鼠淋巴细胞发生凋亡,具有潜在的抗炎免疫调节活性。
Objective To investigate the effects of Cucurbitacin B (Cucurbitacin B) on lymphocyte activation, proliferation, apoptosis and IFN-γ expression in mice induced by polyclonal mitochondria and to explore the mechanism of its immunoregulation. Methods After CuB-treated mouse lymphocytes were stimulated with concanavalin (ConA, 5μg / ml) or phorbol ester (PDB, 10-7 mol / L) and ionomycin (Ion, 0.5μg / ml) , The cell count was used to observe the inhibitory effect of CuB on the proliferation of mouse lymphocytes; the expression of CD69 and IFN-γ was analyzed by flow cytometry; the changes of subduploid peak and mitochondrial membrane potential; and the activation level of Caspase 3 was analyzed by immunoblotting. Results The cell count analysis showed that CuB inhibited the proliferation of mouse lymphocytes in a dose-dependent manner. Analysis of the cellular DNA content showed that as the CuB concentration increased, the number of cells at the sub-diploid peak (ie apoptosis peak) increased. Similarly, the expression of early-activated markers CD69 and IFN-γ in CD3 + T cells after CuB treatment were dose-dependently inhibited. The results of JC-1 staining showed that 10μmol / L CuB significantly decreased the mitochondrial membrane potential of mouse lymphocytes (P <0.01). Immunoblot analysis showed that Caspase 3 was activated obviously after treated with 10 μmol / L CuB for 24 h. Conclusion CuB can significantly inhibit the proliferation and activation of lymphocytes in mice and induce the apoptosis of mouse lymphocytes cultured in vitro with potential anti-inflammatory and immunomodulatory activity.