| Objective:1. To study the distribution of C936 T Polymorphism at 3’- untranslated region of human vascular endothelial growth factor(VEGF) gene in Chinese Han Population.2.To investigate the association of the Polymorphism of VEGF 936C/T and the level of Plasma VEGF with diabetic retinopathy(DR).Methods:1.collection and storation of samples, measurements of clinical characteristics All clinical characteristics such as age, gender were collected from all subjects, height,weight and blood Pressure were measured and body mass index(BMI) were calculated.Then they all received a routine examination of Urine Analysis, electrocardiogram, fundus fluorescein angiography, nerve conduction velocity and abdominal ultrasonic inspection.After an overnight fasting, 5ml venous blood sample were taken from the elbow vein.Fasting and Postprandial blood glucose were measured by glucose oxidase method, and Hb A1 c was analyzed by low Pressure liquid chromatography. TC, LDL-C, Bun, Cr and ALT were detected with automatic biochemistry analyzer and the concentration of serum insulin(or C Peptide)and UAER of all subjects was measured by radioimmunity method and chemoluminescence method respectively.For human genomic DNA extraction, EDTA(1.5mildigram EDTA Per milliliter blood) solution was added to 2ml whole blood, Plasma was separated and then the sample was frozen and stored at-20℃ until use.2.Determination of the VEGF Polymorphism by PCR-RFLP1) The extraction of genomic DNAHuman genomic DNA was extracted from whole Peripheral blood according to the explanation of the kit bought from Ta Ka Ra Biotechnology Company.2) The amplification of required fragment Human genomic DNA extracted from whole Peripheral blood as template, the required fragment was amplified by polymerase chain reaction(PCR).Each amplification was performed using 400 ng of DNA in a volume of 50μl containing 10 Pmol of each Primers, 4ul deoxyribonucleotide-5’-triphosphates(d NTP,2.5m M respectively), 5μl10Ñ…PCR Buffer and 2 unit of Taq Polymerase. DNA templates were denatured at 95℃ for5 minutes, and then each PCR reaction was subjected to 30 cycles with a temperature cycle consisting of 95℃ for 30 seconds, and 68℃ for 30 seconds and 72℃ for 1 minute, and finally an extension at 72℃ for 10 minutes.3) The analysis of restriction fragment length polymorphism(RFLP).After the gel electrophoresis of the PCR products, the Products which have been successfully amplified were subjected to restriction fragment length polymorphism(RFLP)analysis by digestion with 5 unit of the restriction endonuclease Nla III for 10μl of PCR sample at 37℃ for 3 hours in the buffer, then 5ul product(mixed with 0.5ul gold dye and1 ul loading buffer) was separated by 4% agarose gel electrophoresis. And restriction DNA fragments were scored according to the Patterns of DNA bands. The 936 CC genotype remain uncut, whose resulting fragments were 208 bp,while the 936 TT genotype was cut into two fragment of 122 b P and 86 b P,and the 936 CT genotype has three fragment of208 b P, 122 b P and 86 b P.3.Statistical analyses:All calculations were performed using the SPSS 13.0 version for Windows.Chi-square analysis was used to test whether VEGF genotype distribution followed the Hardy-Weinberg equilibrium. The data were non-normally distributed and values are given as medians(inter quartile range). All other variables were presented as means±SD( x ± s).One-way analysis of variance(ANOVA) or Kruskal-Wallis test was used to compare continuous variables among groups. A Chi-square analysis was performed to compare noncontinuous variables among groups. Multiple regression analysis was used to estimate the relationship between DR and other clinical characteristics. We measured the contribution of the polymorphism and interactive effects with other risk factors for type 2diabetic Retinopathy using multiple Forward Conditional logistic regression analysis.Results:1. All the genotype CC, CT, TT and allele C, T can be identified in all the subjects and there was no significant deviation from Hardy-Weinberg equilibrium for any genotype.The genotype CC and allele C frequency in NPDR group were significantly higher than those in DM group and control group(genotype CC frequency: 68.77%,50.02% and46.69% respectively, allele C frequency: 81.27%,64.50% and 67.52% respectively,P<0.01),but there was no significant difference between in DM group and control group.The genotype CT and allele T frequency in NPDR group were significantly lower than in DM group and controls(genotype CT frequency: 25.02%,36.98%and 41.69% respectively;allele T frequency :(18.77%,31.54%and 32.52%respectively, P<0.05),but there was no significant difference between in DM group and control group. When compared the distribution of genotype TT, the expect counts of more than 20% checks of R×C tables were less than five, then we combined the genotype of T carriers. The frequency of(CT+TT) in NPDR and PDR group was much lower than in DM group and control group,but there was no significant difference between in DM group and control group(NPDR group: 31.27%,PDR group:30.51%,DM group:50.02% and control group: 53.35%).2. When compared the clinical characteristics among different genotype, we combined the genotype TT and CT as one group in order to avoid the statistical deviation resulted from the low frenquency of homozygous TT. There were no statistically difference of mean age, sex, duration of DM, the level of FBG,HDL-C, BMI and blood pressure between in the genotype CC and genotype of T carriers(CT+TT). Compared with genotype(CT+TT) group, the level of PBG(CC:14.74±5.45 mmol/L,CT+TT:12.58±3.18mmol/L), TC(CC: 5.73±1.38 mmol/L, CT+TT: 5.26±1.39 mmol/L),Hb A1c(CC: 8.94±1.94%, CT+TT: 8.20±1.69%),LDL-C(CC: 3.15 ± 0.85 mmol/L,CT+TT:2.85±0.68 mmol/L), Ln AER(CC:3.93±1.89, CT+TT:2.96 ± 1.49) were significantly increased in CC group(P< 0.05), however the level of FCP(CC:353.98±191.02 pmol/L,CT+TT:462.43±272.84Pmol/L)and PCP(CC: 1231.60±563.56 pmol/L, CT+TT: 1774.26± 593.81Pmol/L) were significantly decreased.(P<0.05).3. These was no significant difference in age and sex among the healthy controls group DM group, NPDR group and PDR group groups. There was no significant difference in the levels of blood pressure,plasma glucose,triglyceride, Hb A1 c and BMI among the healthy controls group DM group,NPDR group and PDR group.However,the level of C-Pe tide in NPDR group were lower than in DM group. There was no difference of the morbidity of peripheral neuropathy between the two groups(χ2=1.433,P=0.231).4. The morbidity of DR in genotype CC group(70.91%) was much higher than in genotype TT( 45.47%) and genotype CT(54.07%), but there was no difference between in genotype TT and genotype CT(P>0.05).5. Logistic regression analysis showed that the VEGF gene Polymorphism had negative correlation with the increased risk of DR.Multiple logistic analysis showed that the level of VEGF,Hb A1 c and LDL-C had Positive correlation with the increased risk of DR.Conclusion:1.VEGF C936 T Polymorphism located in the 3’-untraslated region of VEGF was found in individuals of Han nationality from China. CC genotype probably was a genetic marker of susceptible to DR, while TT genotype probably was a protective genotype of DR.2. Allele C probably was a genetic marker susceptible to DR, while allele T probably was a protective gene of DR.3. In addition to the Polymorphism of VEGF936Câ†'T, the level of Hb A1 c,TC,LDL-C may be risk factor for DR. |
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