可能参与调节中枢神经系统病变的新的信号分子:JWA基因对HEK293细胞谷氨酸转运的影响  

Effect of JWA gene on glutamate transport in HEK293 cells:a potential signal molecule associated with malfunction of central nervous system

作　　者：齐泓[1] 李爱萍[1] 周建伟[1] 

机构地区：[1]江苏省人类功能基因组学重点实验室,江苏省应用毒理学重点实验室分子毒理研究室,南京医科大学公共卫生学院,江苏省南京市210029

出　　处：《中国临床康复》2004年第7期1259-1261,T002,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30170812);江苏省高校高新技术产业化项目(JH01-49);国家973重大基础研究规划项目(2002CB512900);国家科技部基础研究前期研究专项(国科基字2001-50)~~

摘　　要：目的:探讨新的微管相关蛋白JWA对谷氨酸转运的调控及其机制。方法:用构建JWA高表达载体转染的HEK293细胞,建立JWA高表达模型;用反义寡核苷酸技术抑制细胞JWA表达,建立JWA低表达模型;用激光共聚焦显微镜检测细胞内Ca2+的含量,用免疫荧光和Westernblot法检测细胞内Jwa蛋白的表达水平。掺入犤3H犦-D,L-谷氨酸为标志物观察JWA基因对细胞摄取谷氨酸的影响。结果:JWA基因高表达的细胞摄取谷氨酸增加;用反义寡核苷酸关闭JWA后,谷氨酸摄取下降。佛波酯可使JWA高表达细胞和对照组未转染细胞谷氨酸转运都增加,但未转染细胞与JWA高表达细胞谷氨酸摄取增加无显著差别。佛波酯还可引起HEK293细胞内游离钙浓度一过性升高。结论:JWA编码的微管相关蛋白可通过非PKC途径增强HEK293细胞谷氨酸转运功能,增强谷氨酸介导的神经兴奋传递,参与中枢神经系统病变,因而是一种可能参与调节中枢神经系统病变的新的信号分子。而佛波酯引起的细胞谷氨酸转运功能增强可能与细胞内钙稳态的改变有关。AIM:To explore the mechanism of JWA,a new microtubule-associated proteins,in the regulation of glutamate transport.METHODS:JWA transfected HEK293 cell was obtained as a model for enhancing the expression of JWA.Antisense oligonucleotide of JWA was used to inhibit the expression of JWA so as to construct a model for down-regulating the expression of JWA.The intracellular calcium content was measured by laser confocal microscopy.The expressed proteins in the cells were determined by immunofluorescence microscopy and Western blot.The effects of JWA on glutamate uptake in the cells were observed by incorporation of tritium-D,L-glutamate.RESULTS:Glutamate uptake in the cells with enhanced expression of JWA was elevated and down-regulated as a result of JWA blocked by antisense oligonucleotide.PMA(phorbol 12-myristate 13-acetate) contributed to the increases of glutamate transport in both of transfected and untransfected cells and independent to the JWA expression levels. PMA also induced a transient boost of intracellular free calcium concentration(i).CONCLUSION:JWA enhances the function of glutamate transport in HEK293 cells,which is not through signal pathway of protein kinase C.It is likely to be a new signal molecule associated with malfunction of central nervous system.The increased glutamate transportation induced by PMA may be unrelated to intracellular JWA expression but associated with the alteration of intercellular calcium homeostasis.

关 键 词：中枢神经系统疾病 信号分子 JWA基因 HEK293细胞 谷氨酸转运 调控机制 

分 类 号：R745.1[医药卫生—神经病学与精神病学]