Diabetic Macrovascular Cloning And Functional Analysis

Objective: We have prepared the diabetic monkey model by the methods of high calorie diet followed with injection low dose streptozotocin and found 216 differential expression genes in diabetic aorta with cDNA array technique. Among those genes, adipophilin was up-regulated by 7.8 folds and was the top up-regulated gene in our aorta cDNA array. Therefore we investigate the effect of adipophilin on genesis of diabetic macroangiopathy in order to retrieval the role of adipophilin in artherosclerosis.Methods: Methods of Real time PCR, Northern blot, Westen blot were used to investigate the expression levels of adipophilin in aorta of type 2 diabetic patients and diabetic mokey. Those methods were also used to investigate the effect of glucose, AcLDL, Pioglitazone and berberine on the adipophilin expression in THP-1 derivated macrophage. The effects of adipophilin on proinflammatory cytokines expression in macrophage were studied by the technique of siRNA and overexpression. We observed the effect of adipophilin on AcLDL uptake in THP-1 derivated macrophage by flow cytometry.Results: Adipophilin was found highly expressed in aorta of type 2 diabetic patients and diabetic mokey which was in accord to that of diabetic monkey aorta cDNA array. We found that adipophilin expression was increased in THP-1 derivated macrophage and human aorta smooth muscle cell in dose dependent manner by AcLDL, Pioglitazone and berberine,but not glucose. Overexpression adipophilin increased the AcLDL uptake in THP-1 derivated macrophage and human aorta smooth muscle cell and inhibite AcLDL-induced TNFa expression in THP-1 derivated macrophage. Conclusion: Adipophilin was highly expressed in diabetic aorta. AcLDL, PPAR γ agonist and berberine could increse adipophilin expression in THP-1 derivated macrophage and human aorta smooth muscle cell. Glucose did not effect adipophilin expression in THP-1 derivated macrophage and human aorta smooth muscle cell. Adipophilin could increase AcLDL uptake in THP-1 derivated macrophage and human aorta smooth muscle cell. Overexpression adipophilin could inhibitAcLDL-induced TNFa expression in THP-1 derivated macrophage and adipophilin might be a compensative response to hyperlipidemia and might play a duplicate role in diabetic macroangiopathy. Part IICloning , protein expression and function studying of 4TM genefrom human aortaObsjective: We have prepared the diabetic monkey model by the methods of high calorie diet followed with injection low dose streptozotocin and found 216 differential expression genes in diabetic aorta with cDNA array technique. Among those genes (ESTs), one EST was down regulated by 4 folds. We cloned the full length cDNA by bioinformatics technique and found that it contained 4 transmembrane domains. We name this gene as 4TM gene. Thus we started functional study on this full length cDNA.Methods: 1) Subclone and bioinformatics technique was used to clone full length 4TM cDNA. 2) To construct prokaryotic expression vector and invert into Bacillus coli to express 4TM protein then purify 4TM protein from Bacillus coli and immune rabbit to produce polyclone antibody. 3) Northern blot technique was used to establish 4TM gene expression profile in human tissues. 4) Construct pEGFP-C3-4TM vector and transfect it to 293 cell line to observe the GFP-4TM fusion protein intracellular location. 5) Realtime PCR technique was used to investigate the effect of glucose and AcLDL to 4TM gene expression in human aorta smooth muscle cell. 6) Western blot technique was used to establish 4TM protein expression profile in human tissues. 7) Immunohistochemical method was used to observe 4TM protein location in human aorta.Results: 1) The full length 4TM gene cDNA which contained a 1035bp ORF was cloned from human aorta. 2) The 4TM polyclone antibody was made by immunerabbit with 4TM protein. 3) It was found that 4TM gene and protein was widelyexpressed in human tissue. 4) 4TM protein was expressed at cell membrane. 5) 4TMgene expression was downregulated in human aorta smooth muscle cell by glucose.6)4TM gene expression was decreased in aorta of type 2 diabetic patients and diabeticmonkey.Conclusion: We cloned a novel gene (4TM gene) which might be a membraneprotein and widely expressed in human tissues. 4TM gene expression was decreasedin aorta of type 2 diabetic patients and diabetic monkey and in human aorta smoothmuscle cell by glucose.