Anti-aging Effect And Mechanism Research Underlying Intravenous Transplantation Of Sirt2Over-expression In Human Umbilical Cord Mesenchymal Stem Cells For Alzheimer’s Disease In Transgenic APP~+Mice

Alzheimer’s disease is a form of age-related central nervous system degenerativediseases,characterized by loss of the ability of learning and memory,space cognitivedysfunction,split personality etc. APP+transgenic mouse models could simulate thecharacteristics of AD well. Researches on mesenchymal stem cell transplantation inthe treatment of APP+transgenic mouse become more and more widely,but themechanisms are still unclear. Sirt2,one of the longevity protein family members,could participate in the regulation of nutrient metabolism,aging and apoptosis viaregulating cell cycle,but its potential as a molecular target for treatment of nervoussystem disorders such as AD, has not been determined.ObjectivesTo research the impact of Sirt2over-expression on human umbilicalcord-derived mesenchymal stem cells (hUC-MSCs) by constructing a recombinantadenovirus containing Sirt2gene in vitro. And,to explore the impact and mechanismsof Sirt2over-expressed hUC-MSCs on delaying aging in APP+transgenic model micein vivo.MethodsHuman Sirt2ORF sequence from hUC-MSCs was generated by RT-PCR andinserted into the shuttle adenovirus plasmid to construct positive recombinant shuttleadenovirus plasmid pAdTrack-CMV-Sirt2; The recombinant shuttle-plasmid waslinearized by PmeI and transformed into the BJ5183competent cells to obtainrecombinant adenovirus vector pAd-Sirt2by homologous recombination withpAdEasy1; Recombinant adenovirus vector pAd-Sirt2was linearized by PacI,andthen we transfected linear pAd-Sirt2into HEK293cells by LipofectamineTM2000to create package recombinant adenovirus pAd-Sirt2; The viral titer was determined byair-dilution method; The aged P15hUC-MSCs were infected with pAd-Sirt2,while nocells infected with adenovirus as the control group. We used CCK-8assay todetermine the cell growth curve,flow cytometry to detect the cell cycle,Western Blotto detect expression of aging-related proteins; Then,P3hUC-MSCs were infectedwith recombinant adenovirus pAd-Sirt2to obtain infection group cells andtransplanted into APP+transgenic model mice. Finally, we used Morris water mazeexperiment to detect the ability of learning and memory of model mice three weeksafter transplantation,TBA method for detection of MDA content in serum andHydroxylamine method for detection of SOD activity in serum, real-time fluorescentquantitative PCR (qRT-PCR) for detection of mRNA expression changes of agingrelated mark gene, and Western Blot for detection of protein expression ofaging-related genes.Results1. The expression of Srit2gene in the P15and P2hUC-MSCs had somedifferences. Compared with the P2cells,Sirt2expression in the P15cells wasdecreased significantly.2. We successfully constructed recombinant adenovirus pAd-Sirt2containingSirt2gene by using recombinant adenovirus packaging system and obtained virussupernatant which titer was about1.8x1011pfu/L.3. After infection with recombinant adenovirus pAd-Sirt2,P15hUC-MSCsexpressed GFP clearly under the fluorescence microscope. CCK-8test found thatover-expression of Sirt2can contribute significantly to cell proliferation; Flowcytometry test showed that the percentage of G0/G1cells was reduced and S phase ofthe cell division cycle lengthens significantly for over-expression Sirt2cell group;Western Blot tests showed that aging-related gene expression at the protein level hasdeclined for over-expression Sirt2cells.4. We transplanted P3hUC-MSCs, which were infected by recombinantadenovirus pAd-Sirt2,into APP+transgenic model mice. Morris water maze experiment showed that, compared with the control group, the ability of learning andmemory was markedly improved for the Sirt2over-expression cells transplantationgroup APP+mice; SOD activity increased and MDA content decreased significantlyin the serum; By qRT-PCR and Western Blot Assay, aging-related gene expression oftissues for the Sirt2over-expression cells transplantation group APP+mice at themRNA and protein levels were significantly changed.ConclusionSirt2gene present different expression in P2and P15hUC-MSCs. Sirt2isnegatively correlated with aging; We successfully constructed and packagedrecombinant adenovirus containing Sirt2gene. Over-expression of Sirt2couldsignificantly extend cell cycle of P15hUC-MSCs and promote cell proliferation andeffectively reduce P15aging-related genes expression in hUC-MSCs cells,whichslowed cell Aging; Then we transplanted the P3hUC-MSCs over-expressed Sirt2gene into APP+transgenic mice. Our results showed that the learning and memoryability of APP+transgenic mice were significantly improved; Compared with thecontrol group,aging related genes, such as P21,P16and P53were significantlyreduced,while PCNA and Sirt2were increased.