| Objective: Renin-angiotensin system (RAS) is the most important system to adjust blood pressure in the body. Angiotensin II receptor type 1 is the important part of Renin-angiotensin system (RAS). Angiotensin II combines main771y with receptor type 1 gene to contract blood tube and adjust the excretion of aldosterone, then adjusts the circulating blood flow of blood pressure and organism. The linkage analysis of gene polymorphism and diseases is the common method to study the molecular biological base of multigenic diseases. And the single nucleotide polymorphism (SNP) is the one that exists most in the gene group. This study is to discuss the association of angiotensin II receptor type 1 gene single nucleotide polymorphism with the essential hypertension, and the cause of disease based on the level of molecular biology.Methods: (1) 192 patients with essential hypertension (except for the patients with diabetes and hyperlipemia) in Shandong were chosen as the hypertension group. 20 cases with hypertension as the samples to select SNP. 192 normal cases without hypertension, diabetes and hyperlipemia were chosen as the control group. There are no statistic differences (P<0.05) in the age, the index of body weight, the fasting blood-glucose, the total cholesterin, LDL cholesterin, HDL cholesterin, urea nitrogen and inosine between the two groups. The systolic pressure in the hypertension groupis 156±18mmHg, while in the control group it is 113±10mmHg (P<0.0l), the diastolic pressure in the hypertension group is 97±12mmHg, while the control group is 74±8mmHg (p<0.01). (2) DNA extraction: DNA is extracted from peripheral white cells using a standard phenol-chloroform method, the DNA concentration was measured through sepharose electrophoresis and standardized the concentration value to 20ng/μL. (3) Designing primer: The AT1 genomic sequence (AF245699) from the database of American National Center of Biotechnology Information and its mRNA sequence (NM-000685) were compared to identify the promoter region, 5' untranslated region, intron region, coding region, and 3' untranslated region. Using Primer 3 software developed at Whitehead Institute, the primers of the promoter region, the exons, flanking introns were given PCR amplification. (4) PCR reaction: The reaction used 25μL reaction system on 96-hole plate, including human genome 20ng, 10 × PCR buffer solution 2.5μL, 25mmol/L MC122μL, 10mmol/L Dntp 0.5μL, 10μmol/L primer 0.8μL, 5u/μL Taq DNA polymerase 0.2μL, through GeneAmp9700 PCR System. "Touchdown" PCR was denatured for 2min at 95 ℃, following denaturing (94℃ ,30s) and extension ( 72℃ ,40s), except that in the first 10 cycles, the annealing temperatures decreased from 63℃ to 58℃ by 0.5℃ a cycle for 60s. In the next 30 minutes, the annealing temperature focused at 58℃, lasting 40 seconds once. One final extension step for 7 min was added to stop all the reactions. The primer of PCR amplification was tested by sepharose electrophoresis.(5) SNP identification and detection The amplified PCR purpose fragment was purified by Wizard PCR Preps DNA Purification Resin (Promega), then ABI-PRISM~? BIG-DYE terminal labeling kit (PE, Foster City, CA) was conducted double sequencing reaction using PCR primer as sequencing primer. The sequencing reaction was denatured for 10s at 96 ℃, annealing (50℃,5s) and extension(60℃,4min) in 30 cycles. The products was precipitated in alcohol, ABI377DNA sequencer was used to perform fluorescent labeling terminal double sequencing, then Polyphred software was used to read sequence and SNP affirmation. (6) SNP genotyping Genes with SNP fragments in 192 cases with hypertension and 192 cases in the control group were amplified anddirectly sequenced with Polyphred software reading the genotype of each sample.(7) Statistic analysis Results were processed by SAS. Differences in the distribution of genotypes between groups were determined by the x~2 test. P<0.05 was considered statistically significant.Results: 11 SNP were detected among ATI gene, G165T and G250T were detect... |
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