醛糖还原酶cDNA转染的CHO细胞株的建立及其基因表达与生化特性

The establishment of CHO cell line transfected with aldose reductase cDNA, its gene expression and biochemical characters

目的建立一内含人醛糖还原酶（ｈＡＲ）ｃＤＮＡ的细胞株，并研究其在高葡萄糖环境中的基因表达及生化特性。方法将全长ｈＡＲｃＤＮＡ基因插入质粒载体ｐｃＤＮＡｎｅｏ中，并将ＡＲ正义、反义构建以及ｐｃＤＮＡｎｅｏ载体本身分别转染至中华仓鼠卵巢（ＣＨＯ）细胞中。用放免法测定这些细胞株在高糖环境中的山梨醇水平。用Ｎｏｒｔｈｅｒｎ杂交检测其ｍＲＮＡ表达，并用分光光度法检测ＡＲ活性。结果有四株ＡＲ正义构建的细胞株显示了高山梨醇水平。随机将其中一株细胞作进一步分析，发现该细胞株具有ＡＲｍＲＮＡ表达，并且在高糖环境中ＡＲ活性亦显著升高。以上升高的山梨醇及酶活性均被ＡＲ抑制剂ＨＯＥ８４３所抑制。结论本研究结果表明已成功地建立了一细胞系统，它为进一步研究ＡＲ在糖尿病并发症发病机理中的作用提供了手段。

Objective To create a cell line containing human aldose reductase (hAR) cDNA and to investigate its gene expression and biochemical characters under high glucose concentrations. Methods The full hAR cDNA was insert into the expression vector pcDNAneo. Cells from Chinese hamster ovary (CHO) were transfected with the AR sense construct, AR antisense construct and pcDNAneo vector alone. Sorbitol level was determined by RIA. AR mRNA expression was measured by Northern blots and AR activity was determined by using spectrophotometrical method. Results Four sense construct clones showed an elevated sorbitol production and one cell line was randomly chosen for further study. Northern analysis showed this cell line expressed hAR mRNA. Furthermore, this cell line had higher AR enzyme activity in response to raised glucose concentration. Increased sorbitol level and AR activity were significantly inhibited by an AR inhibitor HOE843. Conclusion We have established a cell line which would be an appropriate model to study the effect of AR on the pathogenesis of diabetic complication.