| Alzheimer's disease (AD) is a kind of degenerative disease of central nervous system that more happened to old people. Its pathogenesy is still not illuminated up to now, also the therapeutic methods, many hypothesis were proposed, among of them, amyloid cascade hypothesis was extensively approved.Ecto-deposit amyloidβ-protein(Abeta,Aβ) is one of major pathology character of AD, and maybe the key pathogenetic factor of AD. Amyloid-βpeptide is the main ingredient of senile plaques, contains 39-43 amino acids and is produced from amyloid precursor protein by sequential cleavage involvingβ-secretase and y-secretase. Now,we have found several endopeptidases:mainly Neprilysin, Endothelin transferase, insulin hydrolase. And NEP is the most important enzyme in regulating the catabolism of Aβ.Recently the role of AP degradation in Aβhomeostasis has been increasingly recognized, and the variance of quantity and activity of NEP plays a significance role on Abeta deposition.The crucial factor that lead to the Abeta deposition of AD patients may be the expression falling of NEP. So, it is very promising to up-regulate NEP in the therapy of AD. In my study we overexpressed NEP mediated by adenovirus vector in SK-N-SH cells and observe the suppressive effects of NEP on neurocyte apoptosis induced by Abeta.Objective:To investigate the suppressive effects of neprilysin(NEP) on SK-N-SH cell apoptosis induced by neurotoxic amyloid beta peptide(Aβ25-35). Methods:(1) The adenovirus vector including neprilysin(NEP) was transfected into human embryonic kidney 293 cells by using LipofectaminTM 2000 and genarate adenovirus expressing exogenous genes. The recombinant adenovirus was observed by fluorescent microscope and PCR technique was used to detect target gene.(2) Infects the person nerve metrocyte lump cell SK-N-SH cell, and examine the NEP expression by RT-PCR and Western Blot. Infect the endogenous damaged SK-N-SH cell (sweAPP-SK), and examine the degree of degeneration to Aβpreliminarily.(3) Infects the person nerve metrocyte lump cell SK-N-SH cell induced by Aβ25-35. The Cell survival rate was measured by MTT.The cells apoptosis rate and intracellular ROS were measured by flow cytometry. The expression of poptosis-associated genes bax and bcl-2 in cells were detected by RT-PCR and Western Blotting.Results:(1) Ad-NEP and control adenovirus Ad-GFP were successfully constructed. The recombinant adenovirus can infect the person nerve metrocyte lump cell SK-N-SH cell efficiencily and examine the high expression of NEP. The result of Elisa showed that the high expression of NEP can reduce the deposition in extracellular of Aβ.(2) MTT and flow cytometry indicated that the high expression of NEP reduced the cell apoptosis and the level of intracellular ROS induced by AP25-35. RT-PCR and Western Blotting, showed that suppressive effects of NEP on the Aβ25-35 induced apoptosis may be achieved by reducing the expression level of bax the level of intracellular ROS.Conclusion:Models of pathologically injured cells in patients with AD can be got by using a certain dosage of Aβ25-35, and the high expression of NEP can withstand the neural toxicity caused by AP25-35:The nerve cells infected by AD-NEP may reduce the deposition of Aβobviously through the high expression of NEP, inhibit neural cell apoptosis, and increase cell survival rate. The expression of NEP may exert neuroprotective effect on SH-N-SK cells induced by neurotoxic AP25-35, which might associated with the poptosis-associated genes.This research prepare a valuable experimental proof for AD gene therapy targeting NEP.
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