| Objective: Diabetic retinopathy (DR) and diabetic nephropathy (DN) which cause blind and renal failure are major microvascular complications of diabetes mellitus. Pigment epithelium-derived factor (PEDF), a 418-amino acid 50 Kb glycoprotein, is a multifunctional serine proteinase inhibitor. Recent studies revealed that the expression of PEDF was decreased in the eye and renal tissue but increased in the serum. In retina, advanced glucose ends (AGEs) induce the production of hyperoxide and the activation of nuclear factor-κB (NF-κB). PEDF inhibited retinal inflammation by suppressing AGEs. It could also depress the production of extracellular matrix (ECM) significantly by inhibiting the expression of transforming growth factorβ-1 (TGFβ-1) and connective tissue growth factor(CTGF). PEDF delays the development and progression of diabetic microangiopathy through inhibiting inflammation, fibrosis, oxidative stress and promoting the apoptosis of endothelial cell. Single nucleotide polymorphism is the DNA sequence polymorphism caused by single nucleotide mutation and it is the most frequent genetic mutation of human being. The gene of PEDF is 16 kb, locating in chromosome 17p13.1 and its polymorphism in promoter region may affect the gene expression by regulating gene transcription. We analyzed two SNPs in the promoter region of PEDF with 271 Han people in Hebei province in order to investigate the mechanism of PEDF in the development and progression of diabetic microangiopathy.Methods: The study included 271 inpatients with type 2 diabetes. All the subjects were unrelated Han people in Hebei province. They were divided into control group (patients without diabetic microangiopathy group, 105 cases) and microangiopathy group (patients with diabetic microangiopathy group, 166 cases). Patients with diabetic macroangiopathy were excluded according to history, clinical symptom, correlated examination. Height and body weight were measured to calculate body mass index (BMI) and fasting insulin was detectd to calculate HOMA-IR. Fasting triglyeride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, fasting blood glucose, HbA1c and urinary albumin excretion rate(UAER)were also detectd. We select 30 patients from control group and 33 patients from microangiopathy group randomly to measure serum PEDF. Human genomic DNA was extracted from peripheral blood leukocytes of each individual. Genotypes of the rs12150053 and rs12948385 were analyzed by polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method. Statistical analysis was performed using SPSS13.0 software package. A probability level of 5% was considered significant. T-test was used to compare continuous variables and Chi-square analysis was performed to compare noncontinuous variables. The odds ratio (OR) and 95% confidence interval (CI) were calculated using a logistic regression model. The linkage disequilibrium was analyzed by 2LD software and the frequency of haplotype was analyzed by EH software.Results:1 There was no significant difference in sex constituent ratio. The levels of age, duration of disease, TG, LDL-C, BMI and HbA1c in microangiopathy group were significantly higher than those in control group, while the levels of FBG and TC, HDL-C, HOMA-IR had no significant difference between the two groups.2 The concentration of serum PEDF was increased in microangiopathy group. There was significant difference between the two groups (269.2±138.6 pg/ml vs. 199.6±85.4 pg/ml).3 The two SNPs in the control group were in Hardy- Weinberg equilibrium (P>0.05). The distribution of T/T, C/T and C/C genotypes of rs12150053 -790 C/T in the control group were 79.0%, 20.0% and 1.0%, and they were 67.5%, 30.7% and 1.8% in the microangiopathy group. There was no significant difference between the two groups (P=0.099). The frequencies of C and T allele in the control group and microangiopathy group were 87.1%, 12.9% and 82.8%, 17.2%, respectively. There was significant difference between the two groups (P=0.047). Compared with T/T genotypes, C/T+C/C genotype might increase the risk of developing diabetic microangiopathy. The odds ratio was 1.82 (95%CI=1.027~3.221).4 The distribution of G/G, G/A, and A/A genotypes of rs12948385 -358 G/A in the control group were 75.2%, 21.9% and 2.9%, and they were 59.0%, 36.7% and 4.3% in the microangiopathy group. There was significant difference between the two groups (P=0.024). The frequencies of G and A allele in the control group and microangiopathy group were 86.2%, 13.8% and 77.4%, 22.6%, respectively. There was significant difference between the two groups(P=0.025). Compared with G/G genotypes, G/A+A/A genotype might increase the risk of developing diabetic microangiopathy. The odds ratio was 2.108(95%CI=1.228~ 3.619).5 The two SNPs were in linkage disequilibrium. The combined analysis of the two SNPs indicated that the T/G haplotype which was 77.4%, was the most frequent haplotype in the control group. The haplotype contains T at rs12150053 and A at rs12948385 increased significantly in microangiopathy group. It might increase the risk of developing diabetic microangiopathy. The odds ratio was 1.767(95%CI=1.061~2.944).Conclusions:1 The method of rapid - potassium iodide DNA extraction was a simple, quick and economical way to extracte human genomic DNA from peripheral blood leukocytes. It could be applied to genomic DNA study.2 Duration of disease, HbA1c, BMI, TG and LDL-C, might be the risk factor of diabetic microangiopathy.3 The concentration of serum PEDF was significantly increased in microangiopathy group. The change of serum PEDF might be a predict factor for diabetic microangiopathy.4 The rs12150053 -358G/A and rs12948385 -790C/T SNPs of PEDF in the promoter region were associated with diabetic microangiopathy. The C/T+C/C genotype of -790C/T and G/A+A/A genotype of -358G/A increased the risk of developing diabetic microangiopathy. |
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