| Objective:Macroangiopathy is one of the major complication of type 2 diabetes mellitus( DM) and an impotant cause of morbidity and mortality in these patients.The pathological hallmarks of this complication is atherosclerosis. Recent studies have shown that MMP-9 can over degrade the major components of basement membrane of vascular wall-type IVcollagen,which is the first step of vascular disease , therefore MMP-9 plays a key role in the development of atherosclerotic plaque.Accordingly MMP-9 gene has been a major candiate gene associated with atherosclerosis.There is a polymorphism in MMP-9 gene which arises from cytosine(C)to thymidine(T)transition at position 1562bp upstream from the start of transcription.This mutation can affects the transcription activity of promoter and is found to be functionally important. Simvastatin,an HMG-CoA reductase inhibitors the most common used lipid-lowering drug.Most epidemiological study have shown that deyond lipid lowering,simvastatin also has antiatherosc-lerotic effects. Therefore in this study serum MMP-9 of type2 diabetes was measured and MMP-9 genotype was determined in order to elucidate the association of MMP-9 and its C-1562T polymorphism with type 2 diabetic macroangiopathy, which would be helpful to deeply understand the pathophysiological mechanism of type 2 diabetic macroangiopathy. Meanwhile the changes of serum MMP-9 before and after simvastatin treatment was also investigated to explain the mechanism of its protective effects on vascular.Method: According to the standards of diabetic diagnosis and typing put forward by ADA in 1997,a total of 220 unrelated patients with type 2 diabetes were randomly recruited in this study.These patients were further divided into type 2 diabetes with and without macroangiopathy groups.At the same time 110 healthy controls were selected from population for regular physical examination and workers in our hospital.After an overnight fast,7ml blood samples were taken from the elbow vein.For human genomic DNA extraction,2Na-EDTA(fmal 2%) was added to 3ml whole blood. Serum was separated from the remaining 4ml blood and used for measuring biochemical characterises.Serum and whole blood samples were frozen and stored at -20 until use.Simvastatin was administered to 37 diabetic patients with hypercholesterolaemia,5mg,once daily at bedtime on the base of good glucose controlling.After 4 weeks,blood samples were taken toreexamine biochemical characterises.l)Determination of MMP-9 genotype:Human genomic DNA was extracted from whole blood in EDTA.The required fragment was amplified by polymerase chain reaction(PCR).The primers were forward: 5 ' -GCCTGGCACATAGATGGCCC-3 ' and reverse : 5 ' -CTTCCTAGCCAGCCGGCATA-3' The amplication was performed in a 25ul volume containing 100ng of DNA, 1 unit of Taq polymerase ,2.5ul of 10XPCR buffer, dNTP, BSA and primers. Samples were subjected to initial denaturation at 95 for 5 min,followed by 35 cycles of denaturing at 95 for 20s,annealling at 50 for 30s,extension at 72 for 40s and a final step at 72 for 7 min.After being extracted,the PCR products were digested with Sph I at 37 overnight,then electrophoresed in agarose gel and stained with ethidium bromide.Genotype was scored accordimg to the patterns of DNA bands.The retriction site for Sph I is 5'-GCATGC-3',which represents the mutant type T allele and produces two fragments for the TT genotype(247 and 188bp),The wild-type C allele only shows one 435bp band,The TC heterozygote has three bands,with size of 435, 247andl88bp.2)Measurements of clinical characteristics: .Serum level of MMP-9 was measured by enzyme linked immunosorbent assay(ELISA).Lipid,glucose,fasting insulin and HbAlc were also measured in all subjects.Insulin sensitivity index (ISI)was calculated.Height> weight > waist and hip circumferences were measured.Body mass index (BMI)and waist hip ratio (WHR)were calculated.3)Statistical methods:Insulin and Lp(a) ,which distribute nonormally,were analyzed after log transition. All continuous variables were presen...
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