两种检测方法对地塞米松诱导小鼠淋巴细胞凋亡的观察

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,为进一步研究淋巴细胞凋亡对机体免疫调节作用机理及其临床应用提供实验依据.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(DEX)使终浓度为10-5 mol/L,培养24 h后,采用瑞氏染色法光镜下观察凋亡细胞形态,用凝胶电泳法观察其DNA ladder;同时设立不加DEX的对照组及新鲜淋巴细胞对照组.结果光镜观察,淋巴细胞在DEX作用下,可见典型的凋亡形态特征变化,主要表现为细胞核边聚、浓缩,以"齿轮状"细胞改变多见;DNA凝胶电泳图谱呈现典型的凋亡DNA ladder,两种方法结果相符.而未加DEX对照组凋亡细胞明显偏少,新鲜淋巴细胞对照组未见凋亡改变.结论 体外条件下用DEX可高效诱导小鼠脾淋巴细胞凋亡,用瑞氏染色法及DNA凝胶电泳法能准确、快速鉴定凋亡淋巴细胞.

Observation of the Mouse Lymphocytes Apoptosis Induced by Dexamethasome with Two Methods

Objective To explore an empirical method that apoptosis of lymphocytes of mice spleen was induced by dexamethasome(DEX) and detected in vitro,and to provide some empirical evidences for advanced research about action of apoptotic lymphocyte on immunological regulation and clinical application.Methods The lymphocytes were isolated from mice spleens.The experimental group was induced by DEX at a final concentration of 10-5 mol/L,and the control group was not induced.During different incubating periods,the lymphocytes were detected by DNA gel electrophoresis and Wright staining.The fresh cell group was not cultured.Results The typical apoptotic morphological character of lymphocytes,induced by DEX,of the experimental group,was detected under optical microscope.The apoptotic cell was characterized by chromatin condensation which gathered under the nuclear membrane,and the petaloid ambulacra cell was often detected.The DNA ladder was detected by DNA gel electrophoresis in experimental group.It was conformity with the results by Wright staining.The amount of apoptotic cell of control group was smaller than that of experimental group.The apoptotic cell was not detected in fresh cell group.Conclusion The apoptosis of lymphocytes of mice spleen could be induced efficiently by DEX in vitro,and it could be identified accurately and rapidly by Wright staining and gel electrophoresis.