垂体腺苷环化酶激活肽对原代培养海马神经元氧化损伤的保护作用  

Protective effect of pituitary adenylate cyclase-activating polypeptide on oxidative damage in the primary cultured hippocampal neurons

作　　者：崔旭[1] 刘宪霜[1] 房征宇[1] 韩志涛[1] 李文彬[1] 王鲁宁[1] 

机构地区：[1]解放军总医院老年医学研究所,北京市100853

出　　处：《中国临床康复》2005年第45期70-73,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家重点基础研究发展规划项目(G2000057005)~~

摘　　要：目的:在原代培养的大鼠海马神经元上观察不同浓度垂体腺苷环化酶激活肽-38对活性氧所致神经元氧化损伤的保护作用,为垂体腺苷环化酶激活肽-38的神经保护作用提供依据。方法:实验于2001-09/2002-03在解放军总医院老年医学研究所神经生物学研究室完成。取新生1～3d的SD大鼠海马进行神经元原代培养,将细胞随机分成5组:对照组,活性氧组,1×10-8mol/L,1×10-10mol/L和1×10-12mol/L垂体腺苷环化酶激活肽-38组。分别用次黄嘌呤(mmol/L)/黄嘌呤氧化酶(U/L)1.0/200,1.5/150,2.0/200,2.0/300,3.0/300处理细胞诱导氧化损伤模型,选择次黄嘌呤2.0mmol/L/黄嘌呤氧化酶200U/L作为最终的活性氧浓度,进一步观察垂体腺苷环化酶激活肽-38的神经保护作用。采用四甲基偶氮唑蓝比色法测定神经元存活率,光学显微镜下照像,利用计算机图像分析软件分析神经元的形态改变,应用荧光素JC-1染色-激光流式细胞仪检测细胞线粒体膜电位,利用反转录-聚合酶链反应法半定量天冬氨酸特异性半胱氨酸蛋白酶-3的mRNA水平。结果:①细胞存活率分别为:对照组77.8%,次黄嘌呤1.0mmol/L/黄嘌呤氧化酶200U/L活性氧组62.5%,次黄嘌呤1.5mmol/L/黄嘌呤氧化酶150U/L活性氧组50.1%,次黄嘌呤2.0mmol/L/黄嘌呤氧化酶200U/L活性氧组48.4%,次黄嘌呤2.0mmol/L/黄嘌呤氧化酶300U/L活性氧组25.3%,次黄嘌呤3.0mmol/L/黄嘌呤氧化酶300U/L活性氧组28.9%。与对照组比较,神经元的存活率随着培养液中活性氧浓度的增加而呈显著下降趋势(P<0.01)。②活性氧不仅能引起培养神经元的存活率下降(P<0.01),还能导致神经元出现神经退行性变样的形态学损伤,包括细胞皱缩,突起脱落、突起数减少[对照组(2.6±0.6)个,活性氧组(0.5±0.2)个,P<0.01],多极神经元百分率下降(对照组为32.6%,活性氧组为10.1%,P<0.01),同时,也能引起神经细胞的线粒体膜电位显著下降(对照组为40,活性氧组为12AIM：To investigate the protective effect of different concentrations of pituitary adenylate cyclase-activating polypeptide-38（PACAP38） on oxidative damage induced by reactive oxygen species in primary cultured hippocampal neurons of rats so as to provide bases for the neuronal rescue of PACAP38. METHODS：The experiment was conducted in the Department of Neurobiology, Institute of Gerontology and Geriatrics, General Hospital of Chinese PLA from September 2001 to March 2002.The primary cultured hippocampal neurons isolated from the neonatal 1-3 days SD rats were randomly divided into five groups：control group, reactive oxygen species （ROS） group,1×10^-8 mol/L,1×10^-10 mol,/L and 1×10^-2 moL/L PACAP38 groups.The neurons were, treated with hypoxanthine （HX, mmol/L）-xanthine oxidase（XO,U/L）1.0/200,1.5/150, 2.0/200, 2.0/300, 3.0/300 respectively to induce the oxidative damage and choosing HX 2.0 mmol/L-XO 200 U/L as the final concentration of ROS to further investigate the protection effect of PACAP38.The survival rate of neurons was detected by Diphenyl tetrazolium bromide （MTT） colorimetric method. The neuronal degenerative pathological changes were observed by digital picture taken by digital camera connected to the optical microscopy and were calculated and analyzed by means of the computer image analysis software.The mitochondrial membrane potential （MMP） of neuron was labeled by 5,5＇6, 6＇-tetrachloro- 1,1＇,3,3＇-tctrae thylbenzimidazolyl carboeyanine iodine fluorescent and detected by laser flow cytometry.The reverse transcriptionpolymerase chain reaction （RT-PCR）was used for semi-quantitative analyzing the mRNA level of cysteinyl aspartate-speeific protease 3 （caspase-3）. RESULTS：（1） The neuronal survival ratio showed a significantly decreased tendency with the increase of ROS generation concentration in the cultural medium when compared with control group （P 〈 0.01）.The celt survival ratio in deferent groups was：control group 77,8%,1.0/200 ROS gro

关 键 词：神经元 肽类 活性氧 体外研究 

分 类 号：R338.1[医药卫生—人体生理学]