The Relationship Between Cholesteryl Ester Transfer Protein And Type 2 Diabetic Macroangiopathy And The Effect Of Atorvastatin Treatment

Objective:Marcoangiopathy is one of the major complications of type 2 diabetes mellitus(DM) and an important cause of morbidity and mortality in these patients.The pathological hallmark of this complication is atherosclerosis(As).Type 2 diabetic patients are at high risk of atherosclerotic disease.The increased risk can be partly accounted for by the lipoprotein disorders linked to insulin resistance.Cholesteryl ester transfer protein(CETP),which facilitates the exchange of triglycerides(TG) and cholesterol esters(CE) between lipoprotein particles in humans,is a key regulating factor of lipid metabolism and may therefore alter the susceptibility to atherosclerotic vascular disease in diabetic subjects.Recently,the B1B2 polymorphism of intron 1 of the CETP gene(present or absence of a TaqⅠBrestriction site) was show to be a determinant of HDL cholesterol,therefore CETP plays a key role in the development of atherosclerotic plaque.Accordingly,CETP gene has been a major candidate gene associated with type 2 diabetic macroangiopathy.Therefore,in this study serum CETP of type 2 diabetes was measured and CETP genotype was determined in order to elucidate the association of CETP and TaqⅠB polymorphism with type 2 diabetic macroangiopathy,which would be helpful to deeply understand the pathophysiological mechanism of type 2 diabetic macroangiopathy.Methods:A total of 226 unrelated patients with type 2 diabetes were selected randomly for the present study from among patients who admitted to the third affiliated hospital of HeBei Medical University from January to October in 2002.The diagnosis and assort of diabetes was based on American Diabetes Association(ADA) criteria.These patients were further divided into type 2 diabetes with and without macroangiopathy groups.At the same time 96 healthy controls were selected from population for regular physical examination and workers in our hospital.After an overnightfast,5ml blood samples were taken from the elbow vein.For human genomic DNA extraction,EDTA was added to 1ml whole blood.Serum was separated from 4ml blood and used for measuring biochemical characteristics.Serum and whole blood sample were frozen and stored at -20℃ until use.⑴Measurements of clinical data:The level of serum CETP was measured by enzyme linked immunosorbent assay (ELISA).Blood lipid,fasting glucose,insulin and HbA1C were also measured in all subjects.Insulin sensitivity index(ISI) was calculated.Height and weight were measured and body mass index(BMI) were calculated.⑵Determination of the CETP TaqIB polymorphism: Human genomic DNA was extracted from peripheral blood leukocytes by regular methods.The required fragment was amplified by polymerase chain reaction(PCR).Each ampliation was performed using 300ng of DNA in a volume of 25μl containing 10pmol of each oligonuleotide,0.2 mmol/L deoxyribonucleotide-5'-triphosphates(dNTP), 1.5 mmol/L MgCl2,10mmol/L Tris pH 8.4 and 0.5 unit of Taq polymerase.DNA templates were denatured at 95℃ for 5 minutes, and then each PCR reaction was subjected to 30cycles with a temperature cycle consisting of 95℃ for 30 seconds,and 65℃ for 30 seconds and 74℃ for 45 seconds, and finally an extension at 72℃ for 5 minutes.The PCR products were subjected to restriction enzyme analysis by digestion with 5 unit of the restriction endonuclease TaqⅠB for 10μl of PCR sample at 65℃ for 2 hours in the buffer recommended by the manufacturer and the fragments separated by polyacrylamide gel electrophoresis.After electrophoresis,the gel was treated with ethidium bromide for 10 minutes and DNA fragments were visualized by invisible illumination.The resulting fragments were 174bp and 361bp for the B1 allele and 535bp for the uncut B2 allele.⑶Statistical analyses:All computations were carried out with the SPSS 10.0 version for Windows.All continuous variables were presented as .Chi-square analysis was used to test whether CETP genotype distribution followed the Hardy-Weinberg equilibrium.One-way ANOVA and Nonparametric Tests were u...