Expression of recombinant adeno-associated virus in the brain of rats with a focal embolic stroke via carotid artery  

高滴度重组腺相关病毒载体携带的报告基因经颈动脉对缺血损伤后鼠脑组织的表达(英文)

作　　者：韩宗超[1] 张苏明[1] 李宏伟[2] 阮旭中[1] 肖萧[3] 王涛[2] 汪道文[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030 [2]华中科技大学同济医学院附属同济医院内科.基因研究中心,武汉430030 [3]美国匹兹堡大学基因研究中心

出　　处：《Chinese Medical Journal》2002年第8期1170-1174,149-150,共5页中华医学杂志（英文版）

基　　金：ThisstudywassupportedbythegrantforKeyProjectofNationalNaturalScienceFoundationinChina (No39730170)andthegrantforGeneralProjectofNSFC (No 3 9770 810 )

摘　　要：OBJECTIVE: To study whether recombinant adeno-associated virus (rAAV)　mediated foreign gene, LacZ, could pass the blood　brain barrier by intra-carotid artery delivery and express in vivo in ischemic brain of the focal embolic stroke rats to investigate a possibility of delivering foreign gene through carotid artery to treat acute ischemic stroke. METHODS: The carotid artery territory in 41 rats was embolized with or without arterial-like fibrin rich clots to make a model of focal embolic stroke rat. rAAV containing LacZ gene (rAAV-LacZ) was constructed in 293 cells by calcium phosphate cotransfection. The rats were assigned to one of the following treatments: 1 control (without embolism) groups, including PBS treated (n = 6), pLacZ treated (n = 6 ) and rAAV-LacZ treated (n = 6): 2 embolic groups, including embolism + PBS (n =7),embolism + pLacZ (n = 8) and embolism + rAAV-LacZ (n = 8). Brains were cryosectioned and kappa-Gal stain was performed at 2, 4, and 8 weeks, respectively, after transfection, and then infarct volume was measured and the percentage of LacZ staining-positive cells was calculated. RESULTS: In all the control groups and embolism + PBS treated animal, no kappa-Gal staining-positive cells were found, but in embolism + pLacZ (n = 8) and embolism+rAAV-LacZ groups a lot of kappa-Gal staining-positive cells were found. The expression cells were in the tissues around the infarction. The gene expression persisted only nearly four weeks in embolic group with pLacZ. In the embolic group with rAAV-LacZ the expression was very stable during the experiment course (eight weeks) and the percentage of the expressed cells was significantly higher than that of its contralateral areas at the same time points, respectively. CONCLUSIONS: The plasmid vector and rAAV could enter the brain through the ischemia-damaged blood barrier and foreign gene can be expressed in brain. The positive gene expression is mainly in the peripheral areas of the infarction. rAAV as a permanent expression vector may ultimately be us目的　构建了一种新型的腺相关病毒载体并将其携带的报告基因 (rAAV LacZ)注入了自体血血栓卒中大鼠颈内动脉 ,观察缺血脑组织是否表达外源基因方法　运用钙磷共转染的方法构建rAAV LacZ 4 1只大鼠被随机分为 6组。对照组 :组 1:PBS注射 (n =6 ) ;组 2 :pdx11 lacz注射 (n =6 ) ;组 3:rAAV LacZ注射 (n =6 )。栓塞组 :组 1:脑栓塞 +PBS注射 (n =7) ;组 2 :脑栓塞 +pdx11 lacz注射 (n =8) ;组 3:脑栓塞 +rAAV LacZ注射 (n =8)。在 2 ,4 ,8周动物被处死 ,脑组织经冰冻切片 ,X Gal染色 ,脑梗塞体积和基因阳性细胞表达率分别被计算。结果　所有对照组和栓塞组 1未发现有阳性X Gal细胞表达 ,栓塞组 2和组 3阳性细胞表达主要位于梗塞周边区。Pdx11 lacz表达持续接近 1月时间 ,rAAV LacZ稳定高表达超过 2月 ,梗塞周边区阳性细胞表达率与其对侧同样区域脑组织阳性细胞表达率比较有显著性差异。结论　当血脑屏障受破坏或脑动脉血流受阻时 ,rAAV LacZ可以进入脑组织百年个表达外源基因。阳性基因表达主要位于梗塞周边区。

关 键 词：Gene Therapy Animals Blood-Brain Barrier BRAIN Carotid Arteries Cerebrovascular Accident DEPENDOVIRUS Genetic Vectors Intracranial Embolism Male RATS Rats  Sprague-Dawley Research Support  Non-U.S. Gov't 

分 类 号：R743[医药卫生—神经病学与精神病学]