电压依赖性氯通道在3-吗啉斯德酮亚胺诱导的大鼠海马神经元凋亡中的作用  

Role of voltage-dependent chloride channels in hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine in rats

作　　者：常全忠[1] 张淑玲[1] 尹金宝[1] 

机构地区：[1]遵义医学院珠海校区生理学教研室,广东珠海519041

出　　处：《中国药理学与毒理学杂志》2010年第1期8-12,共5页Chinese Journal of Pharmacology and Toxicology

基　　金：贵州省科技厅资助项目(20072107);贵州省科技厅攻关项目(20093075)~~

摘　　要：目的探讨氯通道与缺血性脑损伤的关系。方法离体培养12d的SD大鼠海马神经元,分为4组:正常对照组、3-吗啉斯德酮亚胺(SIN-1)处理组(SIN-11.0mmol.L-1作用18h)、SIN-1+4-4-二异硫氰茋-2,2′-二磺酸组(DIDS,0.1mmol.L-1作用18h)及SIN-1+4-乙酰氨基-4′-异氰酸茋-2,2′-二磺酸组(SITS,0.5mmo.lL-1作用18h)。DNA荧光染色观察神经元形态及检测凋亡数目的变化;免疫荧光染色观察神经元两种电压门控氯通道(ClC-2/ClC-3)的表达;全细胞膜片钳技术记录神经元氯通道电流。结果Hoechst33258染色显示,正常对照组、SIN-1处理组、SIN-1+SITS组和SIN-1+DIDS组神经元凋亡百分数分别是:(18.61±0.59)%,(50.43±0.56)%,(23.37±0.52)%和(23.37±0.84)%;两种氯通道ClC-2/ClC-3在正常神经元上存在;SIN-1处理后的神经元氯通道电流较正常增加55%～56%;氯通道阻断剂SITS和DIDS分别能抑制氯通道电流的30%～40%和50%～60%。结论电压依赖性氯通道可能参与了SIN-1诱导的神经元凋亡,氯通道可能参与缺血性脑损伤过程。OBJECTIVE To explore the effect of chloride channels on the neuronal injury following cerebral ischemia.METHODS Tweleve day in vitro（12dIV） neurons in rats were randomly divided into normal control,3-morpholinosydnonimine（SIN-1,1.0 mmol·L^-1 for 18 h） group,SIN-1＋4-acetamido-4＇-isothiocyanatostilbene-2,2＇-disulphonic acid（SITS,0.5 mmol·L^-1） group and SIN-1＋4,4＇-diisothiocyanatostilbene-2,2＇-disulfonic acid（DIDS,0.1 mmol·L-1） group.Drugs were added with SIN-1 simultaneously and coincubated for 18 h.The neuronal apoptosis and morphological changes were detected with Hoechst 33258.Chloride channels（ClC）-2/ClC-3 were analyzed with immunofluorescence,the chloride channel currents were recorded with whole cell patch-clamp technique.RESULTS Hoechst 33258 staining showed that the apoptotic percentages were（18.61±0.59） %,（50.43±0.56）%,（23.37±0.52）% and（23.37±0.84）% in normal control group,SIN-1 group,SIN-1＋SITS or SIN-1＋DIDS groups,respectively.ClC-2/ClC-3 were positively expressed in normal neurons.The currents in neurons exposed to SIN-1 were increased about 55%-56%,SITS and DIDS,two kinds of chloride channel blockers could inhibited the currents about 50%-60% and 30%-40%,respectively.CONCLUSION Voltage-dependent chloride channel maybe participate in the neuronal apoptosis induced by NO,and the activities of chloride channels are perhaps involved in the cerebral ischemic injury.

关 键 词：氯通道 神经元 脑缺血 海马 膜片钳技术 全细胞 

分 类 号：R966[医药卫生—药理学] R338.8[医药卫生—药学]