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目的 检测AR基因 5调控区可能引起启动子功能及蛋白表达的基因改变。方法 应用PCR SSCP对AR基因 5’调控区进行筛选 ,所有变异体均进行DNA序列分析并克隆至氯霉素乙酰转移酶报告基因载体 (pCAT) ,然后转染Hela细胞 ,检测CAT活性求出启动子相对活性。同时进行凝胶滞留试验与足纹分析以确定DNA与蛋白质的相互作用。结果 在 145名 2型糖尿病患者及 12 3名正常对照的醛糖还原酶基因 5’调控区除野生型外 ,发现两种多态性C( 10 6 )T和C( 12 )G。在正常对照和 2型糖尿病患者中 ,基因型WT/WT ,WT/C( 12 )G和WT/C( 10 6 )T的发生率无显著性差异 在有视网膜病变和无视网膜病变的 2型糖尿病患者中 ,WT/C( 10 6 )T的发生率分别为 31 5 %和17 5 %(P <0 0 5 ) ,WT/C( 12 )G的发生率分别为 10 5 %和 2 5 %(P >0 0 5 ) ,在有并发症的患者中 ,WT/C( 12 )G和WT/C( 10 6 )T的总发生率为 4 1 8%,明显高于无视网膜病变患者的发生率 2 0 0 %(P <0 0 2 5 ) ,野生型 ,C( 12 )G和C( 10 6 )T的相对转录活性分别为 15 7%,31 0 %和 32 2 %,DNA 蛋白质相互作用实验表明 ,这些变异未改变DNA与反式作用因子的结合位点。结论 在中国人群中 ,AR基因 5调控区C( 10 6 )T和C( 12 )G多态与 2型糖尿病视网
Objective To detect the gene changes that may cause promoter function and protein expression of AR gene 5 regulatory region. Methods The 5 ’regulatory region of AR gene was screened by PCR SSCP. All the variants were sequenced and cloned into the chloramphenicol acetyltransferase reporter gene vector (pCAT), then transfected into Hela cells to detect the activation of CAT Sub-relative activity. Gel hold-up and footprinting were also performed to determine the DNA-protein interaction. Results There were two polymorphic C (10 6) T and C (12) G polymorphisms in the 5 ’regulatory region of aldose reductase gene in 145 patients with type 2 diabetes mellitus and 12 3 normal controls. There were no significant differences in the incidence of genotypes WT / WT, WT / C (12) G and WT / C (10 6) T between normal controls and type 2 diabetic patients. In type 2 diabetic patients with retinopathy and without retinopathy The prevalences of WT / C (10 6) T in diabetic patients were 31 5% and 17 5%, respectively (P 0 05). The incidences of WT / C (12) G were 105% and 25% % (P> 0.05). The overall incidence of WT / C (12) G and WT / C (106) T in patients with complications was 41.8%, significantly higher than those without retinopathy The relative transcriptional activity of wild type, C (12) G and C (10 6) T was 15 7%, 31 0% and 32 2%, respectively. The incidence of DNA was 200% (P 0 02 5) Protein interaction experiments showed that these mutations did not change the DNA and trans-acting factor binding sites. Conclusions In the Chinese population, C (10 6) T and C (12) G polymorphisms of AR gene 5 regulatory region and type 2 diabetes mellitus