【摘要】 背景:作为一种强效血管和神经活性肽,内皮素1在各种中枢神经系统病理生理情况下表达增加,可能对神经组织产生有害作用。然而,增高的内皮素1有无直接诱导神经元凋亡的作用尚不清楚。目的:观察内皮素1有无直接诱导原代培养神经元凋亡的作用。设计:以细胞为研究对象,完全随机对照实验研究。单位:一所大学医院的神经内科和一所大学的病理学教研室、生命科学技术学院组织与移植免疫实验中心。材料:实验于2001-03/2002-02在暨南大学医学院病理教研室和生命科学和技术学院组织移植与免疫研究所实验室进行。来自于新生大鼠大脑皮质的原代神经元培养,新生大鼠由中山大学医学中心动物研究所提供。干预:原代大脑皮质神经元培养5d后分别加入0.2,20nmol/L内皮素1处理24h,用AnnexinV,Hoechst33258染色半定量测定细胞凋亡。再用流式细胞仪分别定量检测内皮素受体A拮抗剂或内皮素受体B拮抗剂对20nmol/L内皮素1诱导神经元凋亡的效果。对照组加入等量磷酸盐缓冲液。主要观察指标:观察内皮素1直接诱导培养神经元凋亡的作用,以及其通过的ET受体亚型。结果:加入0.2nmol/L内皮素124h后,Annexin-V和Hoechst33258阳性染色细胞率分别为(23.00±9.96)%,(9.82±0.95)%,与对照组相比差异无显著性意义犤(Annexin-V:(13.50±3.35)%;Hoe更多还原

【Abstract】 BACKGROUND:Endothelin(ET) 1 is a peptide with potent actions on blood vessels and nerve system.Its expression increases in the central nervous system(CNS) in a variety of pathological conditions, inducing harmful effects on the nervous tissue.However it is not clearly elucidated whether the over expressed ET 1 can directly induce neuronal apoptosis. OBJECTIVE:To investigate whether ET 1 can directly induce apoptosis in primarily cultured brain neurons of rat,and which ET receptor subtype(s) is involved in this action. DESIGN:Completely randomized and controlled experimental study based on cells. SETTING:Neurological department in a university hospital, pathological department of a university and laboratory center of tissue transplantation and immunology,life science and technology college. MATERIALS:This study was completed in the Pathology Department,the Institute of Tissue Transplantation and Immunology,the Life Science and Technology College of Jinan University.The subjects were primarily cultured neurons obtained from cerebral cortex of newborn rats that were provided by the Experimental Animal Center of the Medical College,Sun Yat sen University. INTERVENTIONS:After culturing for five days, the neurons were treated with ET 1(0.2 nmol/L and 20 nmol/L) for 24 hours. Apoptotic neurons were semi quantitatively measured with Annexin V and Hoechst 33258 staining respectively.ET 1(20 nmol/L), with BQ123(a selective antagonist for ET receptor A,1 mmol/L) or with BQ788(a selective antagonist for ET receptor B,1 mmol/L),was added respectively into the cultures simultaneously.And the apoptotic neurons were quantitatively measured with flow cytometry 24 hours later.Equal amount of PBS, instead of ET 1,waw added into the control subjects. MAIN OUTCOME MEASURES:The effect of ET 1 on apoptosis rate of cultured rat cortical neurons,and the ET receptor subtypes involved in this action. RESULTS:Twenty four hours after treated with 0.2 nmol/L ET 1,the Annexin V,and Hoechest 33258 positive stained cell rates[(23.00±9.96)%,(9.82±0.95)%]were of no difference as compared with those of the controls[(13.50±3.35)%,(8.21±2.17)%]. By contrast,after incubation with the higher dose of ET 1(20 nmol/L),significant higher rate of apoptosis was measured in Annexin V staining[(50.50±10.78)%,P =0.01,n=4] and Hoechest 33258 staining[(13.78±1.52)%,P =0.000,n=8].Analyzed with flow cytometry,the apoptosis rate was(0.20±0.15)%in the control group,(26.11±3.28)%in 20 nmol/L ET 1 group,and(13.58±4.92)%in BQ123+ET 1 and(9.99±3.30)%in BQ788+ET 1 respectively, indicating that BQ123 and BQ788 partially blocked the apoptosis effect of ET 1 on cultured neurons(BQ123+ET 1 vs ET 1,P=0.005;BQ788+ET 1 vs ET 1,P=0.001,n=4, respectively). CONCLUSION:The higher dose of ET 1(20 nmol/L) can directly induce apoptosis of primarily cultured cerebral neurons of rats.The effect of ET 1 inducing neuronal apoptosis may be mediated via both ET receptors A and B.更多还原