The efficiency of expressing human neprilysin by using lentiviral vector transduction in neural stem cells / 中华神经科杂志
Chinese Journal of Neurology
;
(12): 17-21, 2013.
Article
in Chinese
| WPRIM
| ID: wpr-431253
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 pg0.08 ±0.01 ;1.0 μg0.04 ±0.01 ;2.5 μg0.05 ±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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Neurology
Year:
2013
Type:
Article
Similar
MEDLINE
...
LILACS
LIS