The Role Of TREM2 Modified Bone Marrow Mesenchymal Stem Cells In The Treatment Of Alzheimer's Disease

Background:Alzheimer's disease(AD)is a neurodegenerative disease characterized by a decline in cognition,memory,and changes in behaviors,which severely affect the patients'quality of life.Cholinesterase inhibitors,N-methyl-D-aspartate receptor antagonists and brain metabolic activators are commonly used in the treatment of AD.However,these drugs can only improve early symptoms in AD patients,but can't prevent its progression and deterioration.Therefore,seeking safe and effective treatment is urgently needed.The deposition of amyloid beta-protein(A?)plays a key role in the progression of the disease.Microglia in the brain can effectively keep the balance between the production and clearance of A? by effectively removing A? in the plaque by activated microglia.A key protein for the activation of microglia is the triggering receptor expressed on myeloid cells 2(TREM2),a receptor for the TYRO Protein Tyrosine Kinase Binding Protein(DAP12).Therefore,the aims of the study were to test how bone marrow mesenchymal stem cells transfected by TREM2 overexpressing plasmid affected the APP/PS1 transgenic mice.The bone marrow mesenchymal stem cells were firstly transfected with TREM2 gene eukaryotic expression plasmid and and then were injected into the lateral ventricle of the APP/PS1 double transgenic mice by stereotactic transplantation.Later,the mice's behaviors were examined with the radial six arm water maze.After that,the quantity and size of A? deposition in frozen section of the mice brain were determined by Sulphur S Staining,the concentration of soluble A? 40 and A?42 in the brain was measured by ELISA.The effects of the bone marrow mesenchymal stem cells,empty plasmids modified bone marrow mesenchymal stem cells and TREM2 modified bone marrow mesenchymal stem cells on the expression of TREM2 and DAP12 in the hippocampus of transgenic mice were examined by fluorescence quantitative PCR and Western blot.The study would provide more experimental evidence and new means for the treatment.Section 1 Transfecting the bone marrow mesenchymal stem cells with TREM2 overexpressing plasmidObjectives:To construct TREM2 overexpression plasmid,and to transfect bone marrow mesenchymal stem cells with TREM2 overexpressing plasmid.Methods:1.After the C57BL/6N mice(1-3 day old,SPF grade)were sacrificed,the femur and tibia were taken out under aseptic conditions,then the bone marrow mesenchymal stem cells were primarily isolated and identified after several subcultures.2.The CDS sequence of TREM2 gene was chosen based on gene bank of the NCBI.The TREM2 overexpression plasmid was constructed and identified by restriction endonuclease digestion.Then,the TREM2 overexpression plasmid was transfected into the bone marrow mesenchymal stem cells,and the cells transfected with pEGFP-TREM2 over expression plasmid were verified by fluorescent quantitative PCR.Results:1.The mouse bone marrow mesenchymal stem cells were small and round 3 days after primary isolation and fusiform 5 days after.The mouse bone marrow mesenchymal stem cells were CD105 positive(over 95%),and CD34 and CD45 negative,indicating the successful separation of the cells.2.The bands of 473 1bp and 698bp in plasmid pEGFP-TREM2 after EcoR?/Hind? double enzyme digestion were obtained,which confirmed that the plasmid pEGFP-TREM2 was successfully constructed.3.Compared with the control group,the expression of TREM2 gene in the TREM2 overexpressing group increased significantly(P<0.05).Conclusions:1.The whole bone marrow adherence screening method can effectively separate bone marrow mesenchymal stem cells.2.pEGFP-TREM2 over expression plasmid was successfully transfected into bone marrow mesenchymal stem cells.3.The bone marrow mesenchymal stem cells transfected by pEGFP-TREM2 overexpressing plasmid can upregulate TREM2 expression.Section 2 The histological and functional study of the effects of bone marrow mesenchymal stem cells transfected by TREM2 overexpressing plasmid on APP/PS1 transgenic miceObjective:To test the effects of bone marrow mesenchymal stem cells transfected by the TREM2 overexpressing plasmid on APP/PS1 transgenic mice.Methods:Twenty four 6-month old APP/PS 1 double transgenic mice were randomly divided into 4 groups(n=6 for each group)and treated with injection(2 ?1)to the right lateral ventricle of:1)bone marrow mesenchymal stem cells,2)bone marrow mesenchymal stem cells transfected with empty plasmid,3)bone marrow mesenchymal stem cells transfected with plasmid,pEGFP-TREM2 4)normal saline;respectively.The behaviors of the mice were examined with six arm water maze after 4 weeks.In the mice's brain tissues,the amount and size of A? deposition were examined by Sulphur S Staining,and the content of soluble A? 40 and A? 42 was quantified by ELISA.In addition,in the moice s hippocampus,the expression of TREM2 and DAP12 mRNA was detected by fluorescence quantitative PCR,and the expression of TREM2 and DAP12 protein was detected by using Western Blot method.Results:1.Compared with the mice in the normal saline group,the expression of APP and PS1 genes in the APP/PS1 double transgenic mice increased significantly(P<0.05),which proved that the APP/PS1 double transgenic mice were in line with the Alzheimer s disease mice model.2.No significant difference in the mice's behaviors was found between normal saline group and the other group at day 1 and 2.Starting from day 3,the incubation period and the error number in theMSCs transfected with pEGFP-TREM2 group decreased significantly(P<0.05).3.The amount and size of A? deposition detected by Sulphur S Staining in the MSCs transfected with pEGFP-TREM2 group were significantly smaller than that in the normal saline group(P<0.05).4.Compared with the normal saline group,the content of A? 40 and A? 42 in the MSCs group,MSCs tranfected with empty plasmid group and MSCs transfected with pEGFP-TREM2 group decreased significantly(P<0.05),and the decrease in the MSCs transfected withpEGFP-TREM2 group was the most obvious.5.Compared with the normal saline group,the expression of TREM2 and DAP12 detected by fluorescence quantitative PCR in the MSCs group,MSCs transfected with empty plasmids group and MSCs transfected with pEGFP-TREM2 group increased significantly in the hippocampus(P<0.05).6.The expressions of TREM2 and DAP12 detected by Western Blot in the the MSCs group,MSCs transfected with empty plasmids group and MSCs transfected with pEGFP-TREM2 group were significantly higher than those in the normal saline group(P<0.05).Conclusions:Application of MSCs transfected by TREM2 overexpression plasmid into the APP/PS 1 double transgenic mice can significantly increase the expression of TREM2 and DAP12 in the brain,reduce the deposition of A? in the brain,and improve the mice's behaviors.