醛糖还原酶抑制剂细胞筛选模型的建立  

An Aldose Reductase Inhibitor Screening Model Constructed by Transfection of pSNAV-AR into HEK293 Cells

作　　者：刘静[1] 刘建伟[1] 杜明梅[1] 杨立娜[1] 叶玲[1] 

机构地区：[1]中国人民解放军总医院老年医学研究所,北京100853

出　　处：《中国生物工程杂志》2009年第9期12-16,共5页China Biotechnology

基　　金：国家自然科学基金(30472172);北京市自然科学基金(7092097)资助项目

摘　　要：目的:将糖尿病慢性并发症相关基因醛糖还原酶(aldose reductase,AR)基因与腺相关病毒(adeno associated virus,AAV)表达载体pSNAV2.0重组,使其在HEK293细胞中表达,以基因工程表达的AR为靶向,建立醛糖还原酶抑制剂(aldose reductase inhibitor,ARI)细胞筛选模型。方法:首先采用酶切、连接等方法构建含有人AR基因序列的AAV表达载体pSNAV-hAR,将重组质粒转染HEK293细胞,通过活性测定、Western blot和免疫荧光检测目的基因转染及表达的情况。结果:PCR、酶切、DNA测序均证实表达质粒pSNAV-hAR构建正确。转染HEK293细胞后,一系列分析结果显示,腺相关病毒表达载体介导的AR真核细胞表达产物是具有功能活性的目的蛋白。应用经典醛糖还原酶抑制剂Sobinil和Zopolrestat对此模型进行了验证。结论:AR高表达细胞模型的建立,为进一步筛选ARI、探讨多元醇通路学说在糖尿病慢性并发症发病机制中的作用奠定了基础。Objective： Aldose reductase, involved in the pathogenesis of diabetic complications, was recombinated with an adeno associated virus vector pSNAV2.0, and it was transfected into human embryonic kidney 293 （HEK 293） cells. The gene engineering produced AR would be used as a target protein to screen aldose reductase inhibitors. Restriction endonuclease digestion and ligation procedures were performed to construct the AR expression plasmid vector pSNAV-hAR. Methods： After confirmation the recombinant plasmid by PCR, restriction endonuclease digestion, and DNA sequencing, pSNAV-hAR was transfected into HEK293 cells. Western blot and immunofluorescence analysis were performed to detect the expression of AR and its enzyme activity. Results： The results of a series of analysis including AR activity assay, Western blot and immunofluorescence analysis shown the expressed protein mediated by the adeno associated virus vector transfecting HEK 293 cells, was functional AR. The traditional aldose reductase inhibitors, Sobinil and Zopolrestat, were used to test and verify the constructed cell model. Conclusion： The established AR expression model can be used in mechanismresearch of activation of polyol pathway on diabetic complications and screening potential aldose reductase inhibitors.

关 键 词：醛糖还原酶 腺相关病毒 细胞模型 

分 类 号：Q789[生物学—分子生物学]