CAJ | 학술논문

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123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(-106)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(-106)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(-106)T 的发生率分别为 31.5%和17.5% (P<0.05), WT/C(-12)G的发生率分别为 10.5%和2.5%（P>0.05）,在有并发症的患者中, WT/C(-12)G 和 WT/C(-106)T的总发生率为41.8%,明显高于无视网膜病变患者的发生率20.0%(P<0.025),野生型, C(-12)G和C(-106)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(-106)T 和 C(-12)G多态与2型糖尿病视网膜病变密切相关。
목적　검측AR기인5조공구가능인기계동자공능급단백표체적기인개변。 방법　응용PCR-SSCP대AR기인5'조공구진행사선，소유변이체균진행DNA서렬분석병극륭지록매소을선전이매보고기인재체（pCAT），연후전염Hela세포，검측CAT활성구출계동자상대활성。동시진행응효체류시험여족문분석이학정DNA여단백질적상호작용。 결과　재145명2형당뇨병환자급123명정상대조적철당환원매기인5'조공구제야생형외，발현량충다태성C(-106)T 화 C(-12)G。재정상대조화2형당뇨병환자중，기인형WT/WT, WT/C(-12)G 화 WT/C(-106)T적발생솔무현저성차이.재유시망막병변화무시망막병변적2형당뇨병환자중, WT/C(-106)T 적발생솔분별위 31.5%화17.5% (P<0.05), WT/C(-12)G적발생솔분별위 10.5%화2.5%（P>0.05）,재유병발증적환자중, WT/C(-12)G 화 WT/C(-106)T적총발생솔위41.8%,명현고우무시망막병변환자적발생솔20.0%(P<0.025),야생형, C(-12)G화C(-106)T 적상대전록활성분별위15.7%, 31.0% 화 32.2%,DNA-단백질상호작용실험표명,저사변이미개변DNA여반식작용인자적결합위점。 결론　재중국인군중,AR기인5조공구C(-106)T 화 C(-12)G다태여2형당뇨병시망막병변밀절상관。
Objective To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P<0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P>0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P<0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.