The efficiency of expressing human neprilysin by using lentiviral vector transduction in neural stem cells
10.3760/cma.j.issn.1006-7876.2013.01.006
- VernacularTitle:慢病毒载体介导的neprilysin基因对小鼠神经干细胞的转导及表达
- Author:
Wen HUANG
;
Xuean MO
;
Chao QIN
;
Jinou ZHENG
;
Zhijian LIANG
;
Daobin CHENG
;
Yunfei WEI
- Publication Type:Journal Article
- Keywords:
Neprilysin;
Neural stem cells;
Lentivirus;
Transfection;
Amyloid beta-protein
- From:
Chinese Journal of Neurology
2013;(1):17-21
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the transduction efficiency of expressing human neprilysin by using a lentiviral vector (Lenti-NEP) in mouse embryonic neural stem cells (NSC) in vitro.Methods Primary NSC were harvested from C57BL/6J pregnant mouse at embryonic day 11.5 and transducted with LentiNEP.Immunofluorescent stainingand Western blot were performed to detect NEP protein expression in NSC.Degradation of amyloid beta 1-40 (Aβ1-40) 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 μg (21.00 ± 2.51) and 1.0 μg (15.00 ± 0.54) NEP on degrading Aβ1-40 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 50 μmol/L phosphoramidon (0.5 pg:0.08 ±0.01 ;1.0 μg:0.04 ±0.01 ;2.5 μg:0.05 ±0.01,t =17.2,51.3 and 14.1,respectively,all P ＜0.01).The hydrolytic cleavage on degrading Aβ1-40 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.
