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为了进一步研究醛糖还原酶 (AR)基因 5′调控区存在的可引起蛋白质表达发生改变的遗传变异及其对糖尿病并发症的影响 ,应用PCR SSCP对中国人 2型糖尿病患者的AR基因 5′调控区进行筛选 ,在两名糖尿病患者中发现一新点突变C- 1 6 7→A ,使AR基因 5′调控区产生一个新的CCAAT盒。含点突变的两名患者尽管患病多年 ,长期处于高血糖状态 ,但无糖尿病并发症发生。而且他们的红细胞中AR活性都很低 ,处于无视网膜病变患者组的下限范围。将含野生型和点突变DNA片段分别克隆至氯霉素乙酰转移酶报告基因载体 (pCAT) ,检测CAT的活性确定野生型和突变型序列的转录活性。同时进行凝胶滞留试验以观测DNA与蛋白质的相互作用。结果显示 :含C- 1 6 7→A的启动子相对转录活性 (5 .7% )明显低于野生型(15 .7% )。凝胶滞留试验中 ,突变序列迁移速率较野生型慢。以上结果说明 ,AR基因 5′调控区C- 1 6 7→A点突变干扰了启动子区顺式作用元件与反式作用因子的结合 ,导致AR基因转录活性降低 ,使患者组织中AR活性下降 ,从而阻止或减缓 2型糖尿病视网膜病变发生发展。
In order to further study the genetic variation in protein 5 ’regulatory region of aldose reductase (AR) gene and its effect on the complication of diabetes, PCR SSCP was used to detect the 5’ Regulatory region screening, found in a new point mutation in two diabetic patients C- 1 6 7 → A, the AR gene 5 ’regulatory region to produce a new CCAAT box. Two patients with point mutations, despite their long illness and long-term hyperglycemic status, had no diabetic complications. And their erythrocytes AR activity are very low, in the group of patients without retinopathy lower limit. The wild type and point mutation DNA fragments were respectively cloned into the chloramphenicol acetyltransferase reporter gene vector (pCAT) to detect CAT activity to determine the transcriptional activity of wild-type and mutant sequences. A gel retention test was also run to observe the DNA-protein interaction. The results showed that the relative transcriptional activity of the promoter containing C 1 6 7 → A (5.7%) was significantly lower than that of the wild type (15.7%). In the gel retention assay, the mutation sequence was slower than the wild type. The above results indicate that the C- 1 6 7 → A point mutation in the 5 ’regulatory region of the AR gene interferes with the binding of the cis-acting element to the trans-acting factor in the promoter region, resulting in a decrease in the transcriptional activity of the AR gene and a decrease in AR activity in the patient’s tissue , Thereby preventing or slowing the development of type 2 diabetic retinopathy.