淫羊藿次苷Ⅱ对小鼠脾淋巴细胞体外增殖的影响及其机制研究

Effects and mechanism of IcarisideⅡ on the proliferation of mice splenic lymphocytes in vitro

目的:研究淫羊藿次苷Ⅱ对正常小鼠脾淋巴细胞体外增殖的影响,并初步探讨其机制。方法:不同浓度的淫羊藿次苷Ⅱ与刀豆素A(Concanavalin A,ConA)体外处理小鼠脾淋巴细胞,观察淫羊藿次苷Ⅱ对细胞增殖的影响,初步确定其发挥作用的最佳浓度;然后以最佳浓度淫羊藿次苷Ⅱ在不同时间点与ConA协同处理细胞,采用细胞增殖-毒性检测试剂盒检测增殖能力;运用流式细胞仪检测细胞周期分布;用荧光定量分析仪检测细胞内游离Ca2+浓度的变化。结果:淫羊藿次苷Ⅱ在10-12～10-9mol/L的浓度范围作用48小时,对细胞增殖有一定的促进作用,呈浓度依赖性。在10-7～10-4mol/L范围出现抑制作用。以10-9mol/L的淫羊藿次苷Ⅱ处理细胞,发现其与ConA在不同时间点促进细胞增殖作用显著优于ConA单独应用;并且促进细胞由G0/G1期进入S与G2/M期,并以第24小时效果更明显;促进细胞内Ca2+浓度升高,效果以第12小时最明显。结论:不同浓度的淫羊藿次苷Ⅱ对小鼠脾淋巴细胞增殖具有促进作用,有浓度、时间依赖性,这种促增殖作用的机制可能与升高细胞内Ca2+浓度,进而加快细胞由G0/G1期进入S与G2/M期有关。

Objective：To study the effects of Icariside Ⅱ（ICSⅡ） on the proliferation of mice splenic lymphocytes in vitro and elucidate mechanism of its immunoregulation effects.Methods：Mice splenic lymphocytes were dealt with different concentration of ICSⅡ and concanavalin A.The effects of splenocyte proliferation was detected by CCK-8 method in order to select the most effective concentration of ICSⅡ.After the selection of concentration,cells were dealt with it at different time points.The effect of cell proliferation was detected by CCK-8 method.Cell cycle distribution was observed by flow cytometry（FCM） also at different time points.The changes of intracellular calcium concentration of cell were measured by fluorescence microplate reader.Results：After cultivation of cells for 48 hours with 9 concentration gradients of ICSⅡ,a range of 10-12-10-9 mol/L ICSⅡ could promote the proliferation of cells.This promotion presented concentration-dependent effects.A range of 10-7-10-4 mol/L ICSⅡ presented the inhibitory effects.ICSⅡ of 10-9 mol/L was found to promote the proliferation of cells at different time points and this effect was better than those with ConA only.FCM tests could reveal the fact that ICSⅡcan promote cells to enter into the mitosis by reducing the percentage of cells which in G0/G1 phases while rising the percentage of cells in S and G2/M phases,with the effect being most marked in 24 h.The results of the tests taken by the fluorescence microplate reader can reveal that the fluorescence value of the ICSⅡ group was increasing and the effect was most marked in 12 h.Conclusion：Different concentrations of ICSⅡcould promot the proliferation of cells,presenting the concentration-dependent and time-dependent effect.The mechanism of this effect might through increasing the calcium concentration in cell plasm and promoting cells cycle to enter into the S and G2/M phases from G0/G1 phases.