| Objective: Diabetic nephropathy is the most common and serious complication of diabetes mellitus and has already been an important cause of end-stage renal failing. It is generally believed that long-standing hyperglycemia, dyslipidosis, dyshaemodynamic and growth factor are involved in the progression of diabetic nephropathy and the hyperglycemia is regarded the basic factor. Strict glycemia control was show to prevent the development and progression of diabetic nephropathy. However, there is a family resemble phenomenon in the diabetic nephropathy development, and both the development and progression of diabetic nephropathy exhibit remarkable variation amongst individual patients. All these demonstrated that the genetic factor play an important role. Vascular endothelial growth factor (VEGF) is closely related to the development of diabetic nephropathy by regulating the structure and function of glomerular endothelial cell, glomerular hyperfiltration, increasing synthesis of extracellular matrix and kidney enlargement. Recent study revealed that the serum VEGF concentrations elevated persistently several years before the onset of persistent microalbuminuria in type 1 diabetes during childhood and patients with increased VEGF serum concentrations have an increased risk to develop persistent microalbuminuria, which suggested that expression of VEGF may be at least in part genetically determined. Consequently, VEGF gene is thought to contribute to the susceptibility to diabetic nephropathy. Several novel polymorphisms in the VEGF gene have recently been identified. Of particular interest is a –634G/C polymorphism occurring in 5'-UTR of the VEGF gene, which has been found to regulate the VEGF serum lever and is a functional mutation. Therefore, in this study serum VEGF of type 2 diabetes was measured and VEGF genotype was determined in order to elucidate the association of VEGF and its –634G/C polymorphism with type 2 diabetic nephropathy, which would be helpful to deeply understand the pathophysiological mechanism of type 2 diabetic nephropathy.Methods: According to the standards of diabetic diagnosis and typing put forward by ADA in 1997, a total of 216 unrelated patients with type 2 diabetes were randomly recruited in this study. These patients were further divided into type 2 diabetes with and without nephropathy groups according to their UAER. At the same time 98 healthy controls were selected from population for regular physical examination in our hospital. After an overnight fast, 5ml blood samples were taken from the elbow vein. For human genomic DNA extraction, 2Na-EDTA was added to 1ml whole blood. Serum was separated from 4ml blood and used for measuring biochemical characteristics. Serum and whole blood sample was frozen and stored at -20℃ until use. (1)Determination of the VEGF polymorphism: Human genomic DNA was extracted from peripheral blood leukocytes of each individual. The required fragment was amplified by polymerase chain reaction (PCR) method. The primers were forward: 5'-ATTTATTTTTGCTTGCCATT-3', and reverse: 5'- GTCTGTCTGTCTGTCCGTCA-3'. Each ampliation was performed using 400ng of DNA in a volume of 50μl containing 15pmol of each oligonuleotide, 4ul deoxyribonucleotide -5'-triphosphates (dNTP), 5μl 10хPCR Buffer and 2 unit of Taq polymerase. DNA templates were denatured at 95℃ for 5 minutes, and then each PCR reaction was subjected to 30 cycles with a temperature cycle consisting of 95℃ for 30 seconds, and 56℃ for 30 seconds and 72℃ for 45 seconds, and finally an extension at 72℃ for 5 minutes. The PCR products were subjected to restriction enzyme analysis by digestion with 1 unit of the restriction endonuclease BsmF1 for 10μl of PCR sample at 80℃ for 3 hours in the buffer and the fragments separated by ployacrylamide gel electrophoresis. After electrophoresis, the gel was treated with ethidium bromide for 10 minutes and DNA fragments were scored according to the patterns of DNA bands. The resulting fragments were 304bp for the uncut C allele and 193bp and... |
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