SDF-1α Decreases The Beta-amyloid Deposition In APP/PS1 Transgenic Mice By Regulating The Chemotaxis Of Microglia

Objective:Bone marrow stem cells have the ability to populate the central nervous system into fully differentiated microglia. Newly differentiated bone marrow-derived microglia express higher levels of proteins that are required for antigen presentation and may thus be more efficient phagocytes than resident microglia. SDF-1 is a chemokine with unique chemotaxis properties on bone marrow progenitors. In this study, we explore whether treatment with SDF-1αcan regulate the chemotoxis of microglia and decrease (3-amyloid deposition.Methods:28-week-old APP/PS1 mice and their wild-type littermates were divided into four groups:WT+PBS, WT+SDF-la, APP/PS1+PBS, APP/PS1+SDF-1α. Animals were given the intracerebroventricular injection weekly with PBS or mouse recombinant SDF-1αfor eight weeks. Microglia and Aβon cerebral cortex and hippocampal region of adult mice were detected by immunofluorescence. The level of Iba-1, the marker of microglia, was detected by western blotting. The weight of the mice was detected weekly during injection.Results:SDF-1 a injection increased the number of microglia in WT mice. Western blotting also showed a similar result. The count and the area of Aβdeposits in SDF-la-injected mice are both less than those in control animals. Quantification analysis showed that a relative abundance of plaque associated microglia was increased by 2.92 and 1.75 fold in the cortex and hippocampus of SDF-1α-treated APP/PS1 transgenic mice than those in control group, respectively. SDF-la-injection had no influence on the body weight of mice. Conclusion:Our results strongly suggest that injecting SDF-la significantly reduced amyloid burden in APPS/PS1 mice. This effect maybe associated with the regulation of the chemotoxis for microglia, especially bone marrow-derived microglia. It is conceivable to propose that SDF-1 may be a safe and promising therapeutic agent for AD treatment. Objective:To construct recombinant adenovirus vector co-expressing SDF-1 (CXCL12) gene, so as to lay a foundation for the subsequent gene therapy.Methods:The recombinant adenovirus vector expressing SDF-1 gene was constructed based on the Adeno-XTM expression system and the mechanism of in vitro ligation. Firstly, the SDF-1 gene fragments were obtained from the plasmid pSDF-1-mCherry and inserted into the pEGFP-N1 vector to get the plasmid pSDF-1-EGFP. Secondly, the plasmid pSDF-1-EGFP and the shuttle vector plasmid pShuttle2 were both digested by the enzymes Nhe I and Not I and then the fragment including EGFP and SDF-1 genes was cloned into pShuttle2 to get the pShuttle2-SDF-1-EGFP successfully. After being verified by enzemy digestion and gene sequencing, the recombinant shuttle vector plasmid was digested by the enzymes PI-Sce I and I-Ceu I and the fragment including EGFP and SDF-1 was cloned into the PI-Sce I/I-Ceu I digested adenovirus skeleton plasmid Adeno-XTM Viral DNA. After being verified by enzemy digestion, the recombinant adenovirus vector plasmid was linearizated and transfected into HEK293 cells by lipofectamine for packaging. The recombinant adenoviruses were further amplificated and purified after PCR identification.Results:The recombinant shuttle vector plasmid were verified by enzemy digestion and gene sequencing. The recombinant adenovirus vector plasmid were verified by enzemy digestion. The expression of GFP could be firstly observed in HEK293 cells on 48h after transfection. The CPE was observed at the 4th or 5th day of amplificaion. PCR identification showed that the adenovirus carried the mice SDF-1 gene..Conclusion:We had constructed recombinant adenovirus vector carrying mice SDF-1 gene successfully, which lay the experimental foundation for the treatment of AD using united gene therapy.