Investigating The Role Of ApoM In Insulin Resistance Signaling Pathway By Constructing ApoM Gene Knockout Mouse Model

Part one:Construction of ApoM gene knockout mouse modelObjective: Crispr/Cas9 technology is an important method for targeted editing cells and animal genomes.In order to investigating the effect of ApoM on glucose metabolism,a heterozygous mouse model of ApoM gene knockout(ApoM^+/–)was constructed by crispr/Cas9 technology.A homozygous mouse model of ApoM gene knockout(ApoM^–/–)was obtained by a mating system of wild type C57BL/6J(ApoM^+/+)mice with ApoM+/-mouse.Methods: The single-guide RNA（g RNA）was designed as the consensus sequence follow ApoM gene exon ATG by crispr/Cas9 system.The Cas9 plasmid was linearized by the Not I site and transcribed and purified separately in vitro by the corresponding kit.The purified sample was diluted and injected into the cytoplasm of the fertilized eggs of C57BL/6J female mice.The treated fertilized eggs were transplanted into the fallopian tube of the same strain of pseudopregnant mice,then the abnormally encoding gene mice(ApoM^+/–)was obtained.Mice tails were snipped to identify the genotype.The ApoM^–/– mice was obtained by a mating system of wild type ApoM^+/+ mice with ApoM^+/– mice and definited according to ApoM RNA,DNA and protein expression,respectively.Results: The ApoM^+/– mice were successfully constructed by Crispr/Cas9 technology,and ApoM-/-mice were obtained by subsequent selective breeding.Conclusion: ApoM gene knockout genetic phenotype,which was created by Crispr/Cas9 technology,can be stably inherited to offspring.Part two: Morphological changes of ApoM gene knockout mice and its effects on insulin signaling pathwayObjective: To observe the histomorphological changes in ApoM gene knockout mice and investigate the effect of ApoM gene on the metabolic pathway of insulin（PI3K,MAPK,AMPK）by ApoM gene knockout technology.Methods: ApoM^+/+ mice and ApoM^+/+ AML12 cells were used as controls.1.The gene expression profile of ApoM-/-mice was analyzed by whole-gene expression profiling.2.The morphological changes of heart,liver,kidney,pancreas,testis,epididymis,fat and lung were observed by optical microscope and electron microscopy microscope.3.ApoM-/-AML12 cells were constructed by crispr/Cas9 technique.The lipid metabolism of ApoM-/-AML12 cells was detected by oil red O staining.4.The protein expression levels and phosphorylation levels of PI3K（upstream proteins IRS1,IRS2,IRS3 and downstream proteins PDK-1,AKT,GSK3）,MAPK（Raf,MAPK,ERK）,AMPK（IRS,Enos,AKT,rab-GTP,AS160）signaling path way were detected by Western blot in ApoM-/-mice and ApoM-/-AML12 cells.Results: 1.Compared with ApoM^+/+ mice,the gene expression profile of ApoM^–/– mice has changed through preliminary analysis of global gene chip expression profile,including 204 pairs up-regulated genes and 204 pairs down-regulated genes.2.Compared with ApoM^+/+ mice,ApoM-/-mice showed significant changes in fatty degeneration and lipid droplets,and a small amount of inflammatory cells were infiltrated.The number of islets was decreased and the islet volume became smaller and the surrounding tissue became unclear,part of the islet atrophy disappeared.Islet cells degenerate edema,islet cells decreased,islets have a small amount of bleeding,pancreatic interstitial congestion.Fat cells volume increased significantly,part of the fat cells fusion.Electron microscopy showed that the accumulation of glycogen was significantly increased,the fat-storing cells were increased significantly,the lipid droplets were increased,the mitochondria were swollen in the liver tissue of ApoM-/-mice.The renal tissue appeared lipid droplets in the renal tubular area,the glomerular podocyte proliferation.There was lipid deposition in the lungs of the lung tissue.2.ApoM-/-AML12 showed intracellular lipid accumulated.3.The expression of P-IRS1/2,IRS4,PI3 K,P-AKT,PDK1 and AS160 protein were decreased;P-c-Raf protein expression was decreased,GSK3β,P-ERK protein expression were increased;ampk protein expression was increased in ApoM-/-mouse liver tissue.Conclusion:1.The structure of the important tissues related glucose and lipid metabolism（liver,pancreas,kidney and lung）were changed significantly,such as lipid and glycogen deposition,fat cells enlargement,and ApoM-/-AML12 showed intracellular lipid accumulated,suggesting that ApoM may be involved in glycolipid metabolism.2.ApoM gene can be involved in glycolipid metabolism by affecting the associated protein expression of PI3 K,MAPK,AMPK signaling pathway.