水通道蛋白4基因沉默与体外培养星形胶质细胞的形态生长及其增殖效应  

Aquaporin-4 gene silencing in the form, growth and proliferative effect of astrocytes cultured in vitro

作　　者：楚燕飞[1] 李兵仓[1] 陈菁[1] 王建民[1] 

机构地区：[1]解放军第三军医大学大坪医院野战外科研究所6室

出　　处：《中国临床康复》2006年第17期88-91,i0006,共5页Chinese Journal of Clinical Rehabilitation

基　　金：军队"十五"指令性课题(01L060)~~

摘　　要：目的:研究短发夹环质粒载体对体外培养的星形胶质细胞水通道蛋白4表达的基因沉默效果。方法:实验于2003-12/2005-05在解放军第三军医大学大坪医院野战外科研究所完成。实验动物为SPF级Wistar新生3d大鼠20只,进行大鼠星形胶质细胞分离、培养。实验使用传至第三代的星形胶质细胞。构建特异性抑制水通道蛋白4表达的短发夹环质粒载体水通道蛋白4RNAipGenesil-2,将体外培养的星形胶质细胞分为水通道蛋白4基因沉默组、水通道蛋白4基因沉默对照组、脂质体组和正常对照组,运用脂质体法转染质粒载体,分别于转染后2,3,5,7d通过观察细胞形态和数量的改变,应用反转录-聚合酶连反应、免疫组织化学分别检测水通道蛋白4mRNA和蛋白的表达,水通透实验验证水通道蛋白4基因沉默后星形胶质细胞的水通透功能。结果:星形胶质细胞水通道蛋白4基因沉默后,水通道蛋白4mRNA及蛋白的表达呈进行性减少,至第7天水通道蛋白4mRNA和蛋白表达水平分别减少了(80.12±1.01)%和(80.2±2.54)%,细胞数量减少约60%,星形胶质细胞从扁平和多角形变成类纤维形胶质细胞。水通透实验证实经水通道蛋白4基因沉默的星形胶质细胞体积增大的速度慢于正常星形胶质细胞。结论:构建的水通道蛋白4基因沉默pGenesil-2质粒载体可抑制星形胶质细胞水通道蛋白4的表达;水通道蛋白4参与星形胶质细胞快速水转运过程;水通道蛋白4也对维持体外培养的星形胶质细胞形态、生长、增殖起重要作用。AIM： To study the gene silencing effect of short hairpin plasmid vector on aquaperin-4 （AQP-4） in astrocytes cultured in vitro. METHODS： This experiment was conducted at the Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA from December 2003 to May 2005. Totally 20 SPF newborn for 3 days Wistar rats were Used and underwent isolation and culture pf astrocytes. Astrocytes of the third generation were used in this experiment. We constructed a short hairpin plasmid vector RNAi pGenesil-2 that could specially suppress AQP-4 expression. Astrocytes cultured in vitro were divided into AQP-4 grnr silencing control group, liposome group and normal control group. Plasmid vector was transferred with lipesome method. We detected the expression of AQP-4 mRNA and protein respectively with reverse transcription-pelymerase chain reaction and immunohistology through observing the change of cellular number and morphology 2,3,5 and 7 days after transfection. Water permeability of astrocytes of AQP-4 after gene silencing was proved by water permeability experiment. RESULTS： After AQP-4 gene silencing, the expression of AQP-4 mRNA and protein presented a progressive reduction, the expression of AQP-4 mRNA and protein was reduced by （80.12±1.01）% and （80.2±2,54）%, respectively on the 7^th day. The cellular number was decreased by about 60%： Water permeability experiment proved that the volume of astrocytes after AQP-4 gene silencling increased slowly compared with normal astrocytes. CONCLUSION： Constructed AQP-4 gene silencing pGenesil-2 plasmid vector can inhibit the expression of AQP-4 in astrocytes; AQP-4 participates in the rapid water transport of astrocyte, and AQP-4 is necessary for sustaining morphology, growth and proliferation of astrocytes cultured in vitro.

关 键 词：小神经胶质细胞 脑水肿 RNA干扰 水通道蛋白 

分 类 号：R742.7[医药卫生—神经病学与精神病学]