下调PTEN基因对缺氧损伤后海马神经元NR2B受体调控机制研究  

The regulation mechanism of shRNAspten to NR2B after neuron subjected to anoxia insult

作　　者：陈力学[1] 刘宝松[2] 石少权[3] 周冀英[1] 桂蓓[1] 邵阳[2] 

机构地区：[1]重庆医科大学附属第一医院实验研究中心、重庆市神经病学重点实验室,重庆400016 [2]第三军医大学大坪医院野战外科研究所三室,重庆400042 [3]中山大学附属第五医院妇产科,珠海519000

出　　处：《重庆医科大学学报》2010年第4期493-496,共4页Journal of Chongqing Medical University

基　　金：重庆市科委自然科学基金资助项目(编号:CSTC,2007BB5287)

摘　　要：目的:探讨用RNAi干扰技术下调第10号染色体同源丢失性磷酸酶张力蛋白基因(Phosphatase and tensin homology deleted on chromosome ten,PTEN)对缺氧损伤引起的神经元NR2B受体表达调控机制。方法:建立海马神经元培养缺氧缺糖(Oxygen-glucose deprivation,OGD)损伤模型,转染shRNAspten-GFP质粒,采用细胞比色分析(Colorimetric assay)法观察神经元膜表面NR2B含量,RT-PCR测定NR2B受体的mRNA的表达,采用Fura2-AM法测定胞内Ca2+浓度,AlamarBlue进行神经元活力分析。结果:shRNAspten-GFP能成功转染到神经元中;细胞比色法显示神经元膜表面有大量NR2B表达,与PshGFP对照组相比shRNAspten治疗组NR2B含量明显降低(P<0.05),RT-PCR测定结果与细胞比色法结果基本一致;OGD损伤组比正常组胞内Ca2+浓度显著升高(P<0.05),转染PTEN基因后胞内Ca2+浓度比PshGFP对照组明显降低(P<0.05);无关对照PshGFP组神经元活力较正常对照组和治疗组均有显著下降(P<0.05)。结论:下调PTEN基因可降低NR2B的表达,阻断Ca2+内流,增加细胞活力,从而保护神经元损伤。Objective：To investigate the regulation of PTEN with RNAi on NR2B after OGD.Methods：In the OGD model with anaerobic gas mixture and deoxygenated,glucose-free extracellular solution,shRNAspten-GFP and unrelated control plasmid PshGFP transfection were done respectively.The quantities of NR2B in the neuronal membrane were quantitatively measured with colorimetric assay,and the expression of NR2B mRNA were analyzed by RT-PCR.The neuronal activity was analyzed by AlamarBlue andintracellular free calcium concentration were measured with Fura-2 AM by laser confocal microscope（LCM）.Results：shRNAspten-GFP plasmid can been expressed successfully in cultured neurons.The study demonstrated that there are numerous NR2B subunit expression on neuronal membrane by colorimetric assay（OPD）assay and RT-PCR.The expression and quantity of neuronal NR2B with shRNAspten-GFP treatment was significantly decrease after oxygen and glucose deprivation（OGD）and reperfusion（P〈0.05）.The neuronal activity with shRNAspten-GFP treatment after OGD was significantly higher than that of PshGFP group（P〈0.05）.But intracellular free calcium concentration with shRNAspten-GFP treatment after OGD was significantly lower than that in OGD group and PshGFP group（P〈0.05）.Conclusion：PTEN down-regulation could effectively decrease NR2B expression level induced by OGD,and then block Ca2＋influx and increase neuronal activity,which protect neuron from anoxia injury.

关 键 词：PTEN shRNAs Fura2-AM AlamarBlue测定 细胞比色分析 

分 类 号：R743.3[医药卫生—神经病学与精神病学]