糖尿病致病基因醛糖还原酶基因及其抑制剂筛选模型的构建及功能研究

目的基因工程方法建立人醛糖还原酶基因(AR)的蛋白分子模型,并将该模型应用于中草药AR抑制剂的初步筛选。方法将含基因AR及融合基因AR∷GFP的重组质粒pcDNA3.1/myc-His-AR及pcDNA3.1/myc-His-AG瞬时转染HEK293细胞,分别命名为HAR、HAG细胞株。通过荧光显微镜观察AR∷GFP的绿色荧光直接判断转染效果及估计基因AR的表达;Western blot、紫外分光光度法检测AR蛋白表达及AR酶活性。应用此HAR模型对黄芩苷等5种中草药进行初步筛选,并与经典AR抑制剂Sorbinil、Zopolrestat的抑制效果比较。结果GFP绿色荧光表达丰富,表明质粒转染效率较高;Western blot和紫外分光光度法显示:转染后HEK293细胞中AR蛋白表达量高,AR酶活性强(空白对照的2.5倍)。AR抑制剂筛选实验揭示,黄芩苷、虎杖苷、蕨麻多糖JM等中草药表现出与Sorbinil相近的AR抑制活性。结论AR的蛋白分子模型成功建立,此模型可得到较大量高活性AR酶蛋白,并可应用于AR抑制剂的初步稳定筛选;黄芩苷、虎杖苷、蕨麻多糖JM等中草药显示出潜在的AR抑制活性。

Construction and function study of a drug-screening model based on aldose reductase gene and its inhibitors

Aim To construct a sensitive and efficient aldose reductase gene-based screening model by genetic engineering method,and to screen the potential effective AR inhibitors from Chinese herbal medicine.Methods The recombinant plasmids(pHAR,pHAG containing AR gene,AR∷GFP chimeric gene) were transfected into the HEK293 cells by liposome-mediated gene transfer method,named HAR,HAG,respectively.The result of transfection was estimated by the green fluorescence under fluorescent microscope straightly.Western blot and UV-spectrophotometry were performed to detect the expression level of AR and its enzyme activity.Meanwhile the model was verified by two aldose reductase inhibitors,Sorbinil and Zopolrestat,as the controls.Five Chinese herbals were tested by the established ARI screening model(HAR).Results The abundant green fluorescence showed the high efficiency of transfection.Western blot and UV-spectrophotometry demonstrated AR expression in HAR abundantly induced by the high glucose concentration.The AR activity increased about 2.5-fold to the negative control.Enzyme kinetics experiments revealed aldose reductase inhibitory activity of the Chinese herbal medicines(baicalin,potentilla anserine polysaccharide JM,and polydatin) were similar to that of Sorbinil.Conclusion A protein molecular model by AR expression in HEK293 cells is established successfully.It can be used for screening the potential AR inhibitors simply and effectively.Chinese herbal medicines baicalin,potentilla anserine polysaccharide JM,and polydatin possess the potential AR inhibitory function.