水通道蛋白4示踪系统的构建及评价  

Construction and evaluation of green fluorescent protein labeled aquaporin 4 tracing system

作　　者：袁志华[1] 姜久昆[2] 潘建[2] 祝建勇[2] 陆远强[2] 

机构地区：[1]安徽省立医院老年医学科,合肥230036 [2]浙江大学医学院附属第一医院急诊科,杭州310003

出　　处：《中华危重症医学杂志（电子版）》2011年第5期5-8,共4页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：浙江省自然科学基金项目(Y205096)

摘　　要：目的构建绿色荧光蛋白(GFP)标识的水通道蛋白4(AQP4)示踪系统,建立表达AQP4的大鼠星形胶质细胞水通透性的评价系统。方法利用基因重组技术,构建pEGFP-C1-AQP4-M23表达质粒,并转染CTX-TNA2星形胶质细胞,利用激光扫描共聚焦显微镜(LSCM)成像技术分析GFP标记的AQP4-M23在细胞上的分布,动态观察钙黄绿素荧光强度随时间的变化,评价AQP4-M23对细胞膜水通透性的影响。结果正确获取AQP4-M23全长序列,利用基因重组技术连接pEGFP-C1质粒和AQP4-M23序列,酶切证实成功构建pEGFP-C1-AQP4-M23质粒。LSCM扫描显示GFP标记的AQP4-M23融合蛋白在细胞膜上均匀分布表达,胞浆内仅痕量表达;表达AQP4-M23的CTX-TNA2细胞对水的通透性明显增加(P<0.01)。结论成功构建了GFP标识的AQP4示踪系统,建立了表达AQP4的大鼠星形胶质细胞水通透性的评价系统,AQP4可使星形胶质细胞对水的通透性明显增加。Objective To construct the green fluorescent protein （GFP） labeled aquaporin 4 （AQP4） tracing system, and establish the water permeability evaluation system for rat astrocytes expressing AQP4. Methods Using the gene recombination technique, the AQPd-M23 sequences were acquired by reverse transcription polymerase chain reaction and authenticated by gel electrophoresis to construct the pEGFP-C1-AQP4-M23. Then the plasmids were transfected transiently into CTX-TNA2 astrocytes which were cultured 24 h to express protein. The laser scanning confocal microscope imaging technology was employed to record fluorescence images of GFP and variation of calcein fluorescence intensity. Results The target gene （AQPd-M23） was acquired, and the restriction enzyme cutting sites of Hind ~ and Xba I were also correctly introduced. The plasmids pEGFP-C1 and the sequence AQP4-M23 were ligated. GFP which located at the amino terminal of AQP4-M23 did not affect the location of AQP4-M23 on cell membrane. Compared with the non-AQP4-M23-expressed CTX-TNA2 astrocytes, the water permeability increased up to 5 folds in the AQP4-M23-expressed CTX-TNA2 astrocytes （P 〈 0.01）. Conclusions In our experiment, tracing system of GFP labeled AQP4 was successfully constructed. The water permeability evaluation system was established for rat astrocytes expressing AQP4. The water permeability increased in the AQP4-M23-expressed astrocytes.

关 键 词：绿色荧光蛋白质类 水通道蛋白质4 通透性 转染 星形细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]