地塞米松诱导小鼠脾淋巴细胞凋亡实验研究

Experimantal Study on Apoptosis of Mouse Lymphocytes Induced by Dexamethasome

目的探讨地塞米松体外诱导小鼠脾淋巴细胞凋亡的实验方法。方法取昆明小鼠,分离其脾脏淋巴细胞制成单细胞悬液,实验组加入地塞米松,使终浓度为10-8、10-6、10-4、10-2mol/L。培养17h,采用流式细胞法观察其凋亡率,凝胶电泳法观察其DNA ladder,蛋白质印迹法观察凋亡基因Bax和抗凋亡基因Bcl-2的表达。同时设立空白对照组和新鲜淋巴细胞对照组。结果流式细胞术观察到给药组凋亡率明显高于正常组,且10-2mol/L的地塞米松组诱导凋亡率最高;DNA凝胶电泳图图谱呈现典型的凋亡DNA ladder;蛋白质印迹分析显示出现凋亡基因Bax和抗凋亡基因Bcl-2相应表达变化。而未加入地塞米松对照组凋亡较少,新鲜淋巴细胞几乎未见细胞凋亡。结论建立诱导小鼠脾淋巴细胞凋亡模型的地塞米松的最佳浓度为10-2mol/L。

Objective To explore the mechanism of spleen lymphocyte apoptosis induced by dexamethasone. Methods Lymphoeytes were separated from spleen ofmiee and divided into experimental group and control group. The lymphocytes in experiment group were treated with dexamethasone at the the final concentration of 10^-8, 10^-6,10^-4,10^-2 mol/L,respectively. 17 h after treatment, FCM was adopted to measured the rate of apoptosis, DNA ladder was used to observe the apoptosis of lymphoeytes, and western blotting was used to detect the expression of Bax and Bcl-2 protein. Results The rate of apoptosis in experiment group was obviously higher than that of control group, and which was highest with dexamethasone at the concentration of 10-4mol/L. DNA gel electrophoresis and western blot revealed typical DNA ladder and expression changes of Bax and Bcl-2 protein after dexamethasone treatment. Conclusion Dexamethasone can induce the apoptosis of mouse spleen lymphocyte in vitro and the metheds of FCM, DNA ladder and western blot can identify the apotosis accurately.