摘要
目的观察慢病毒载体（lentiviralvector）介导的脑啡肽酶（Lenti-脑啡肽酶）基因对小鼠神经干细胞（NSC）转导及其表达的情况。方法利用Lenti-脑啡肽酶基因载体体外转导小鼠原代NSC，细胞免疫荧光染色和蛋白质印迹法分析NSC的蛋白表达，酶联免疫吸附试验和高效液相色谱法检测NSC中脑啡肽酶蛋白对β-淀粉样蛋白l-40（Aβ1-40）的体外降解效率，不同组别之间差异比较采用t检验。结果Lenti一绿色荧光蛋白转导NSC后96h的成功转导率达90％以上。NSC转导脑啡肽酶基因后可同时表达脑啡肽酶和NSC的标记抗体Nestin或神经元的标记抗体微管相关蛋白2。随着细胞脑啡肽酶膜蛋白的浓度增加或孵育时间延长，脑啡肽酶降解Aβ1-40活性也增强。2．5μg（21．00±2．51）及1．0μg（15．00±0．54）脑啡肽酶蛋白降解活性较0．5Ixg（8．004-0．81）显著提高（t=40．4、12．7，均P〈0．01），phosphoramidon则明显抑制脑啡肽酶活性（0．5μg：0．08±0．01；1．0μg：0．044-0．01；2．5μg：0．05±0．01，与相等脑啡肽酶浓度比较，t=17．2、51．3和14．1，均P〈0．01）。孵育1、4和12h的脑啡肽酶降解率分别为11．4％、28．4％和93．7％。结论Lenti-脑啡肽酶基因可于体外成功转导小鼠NSC，移植脑啡肽酶基因转染的NSC具有潜在的治疗前景。
Objective To study the transduction efficiency of expressing human neprilysin by using a lentiviral vector （Lenti-NEP） in mouse embryonic neural stem ceils （NSC） in vitro. Methods Primary NSC were harvested from C57BL/6J pregnant mouse at embryonic day 11.5 and transducted with Lenti- NEP. Immunofluorescent stainingand Western blot were performed to detect NEP protein expression in NSC. Degradation of amyloid beta 1-40 （AI31~0 ） by NEP protein transduced with Lenti-NEP in NSC was analyzed using ELISA and HPLC. Results Over 90% NSC were successfully transduced with Lenti-NEP via observation of fused protein green fluorescent protein under the microscopy. Expressions of NEP transduced with Lenti-NEP in NSC and of the markers of NSC （nestin） and neuron （MAP2）. The enzyme activity of 2. 5 p-g（21.00 ± 2. 51 ） and 1.0 p-g（ 15.00 ± 0. 54） NEP on degrading AI31.40 was shown to improve significantly compared to 0. 5 μg NEP（8.00 ±0. 81 ,t =40. 4 and 12. 7, respectively, both P 〈0. 01 ）. The activity of NEP was inhibited in the presence of 50μmol/L phosphoramidon （0. 5μg ：0. 08 ± 0.01 ;1.0μg： 0. 04 ± 0. 01 ;2. 5μg ： 0. 05 ± 0. 01, t = 17.2,51.3 and 14. 1, respectively, all P 〈 0. 01 ）. The hydrolytic cleavage on degrading A13140 by NEP was 11.4%, 28.4% and 93.7% with incubation for 1 h, 4 h and 12 h, respectively. Conclusions Lentiviral vector successfully delivers NEP gene to NSC in vitro. Targeting on NEP and NSC may provide potential therapeutic tool for Alzheimer＇ s disease.
出处
《中华神经科杂志》
CAS
CSCD
北大核心
2013年第1期17-21,共5页
Chinese Journal of Neurology
基金
国家自然科学基金资助项目（81160152）
广西科学基金资助项目（桂科青0542053）
