氨基酸对胰岛素纤维形成及细胞毒性的影响

Effect of amino acids on formation and cytotoxicity of bovine insulin fibril

目的:研究氨基酸对牛胰岛素淀粉样纤维的生成、形态和细胞毒性的影响.方法:采用硫黄素(ThT)荧光检测牛胰岛素形成淀粉样纤维的动力学曲线,透射电镜观察纤维形态;以淀粉样纤维诱导人红细胞的聚集和溶血为指标评估纤维对细胞膜的损害作用.结果:牛胰岛素在pH1.6,57℃的条件下,可在1.5h开始形成淀粉样纤维,5种氨基酸即组氨酸(His)、脯氨酸(Pro)、苏氨酸(Thr)、谷氨酰胺(Gln)和精氨酸(Arg)具有延迟胰岛素纤维化的作用,同时改变了淀粉样纤维的形态.胰岛素在形成淀粉样纤维后可在较低的浓度范围内诱导人红细胞溶血和聚集,该作用随着纤维的生长逐渐增强.虽然在氨基酸作用下延迟生长的纤维具有不同的纤维形态,但并不能改变纤维对细胞膜的损害作用.结论:牛胰岛素在形成淀粉样纤维后具有破坏细胞膜结构的作用.氨基酸可以抑制胰岛素的纤维化作用.胰岛素纤维对红细胞膜的损害作用与其形态和生长动力学无关.

AIM ： To investigate the effect of amino acids on the formation,morphology and cytotoxicity of amyloid fibril of bovine insulin. METHODS： Fibril formation was monitored by thioflavine T fluorescence and the fibrillar morphology was visualized under transmission electron microscope. The cytotoxicity of insulin fibril was evaluated by measuring hemolysis and aggregation of human red blood cells. RESULTS： Incubation of bovine insulin under acidic medium （pH 1.6） at 57℃for 1.5 h resulted in the formation of amyloid fibril. The growth kinetics and morphology of the fibril changed in the presence of amino acids such as His, Pro, Thr, Gin and Arg. Insulin fibrils induced aggregation and hemolysis of human red blood cells in an aging-dependent man- ner. These effects were not related to the morphology of amyloid fibril. CONCLUSION： Amyloid fibril of bovine insulin has a disruptive effect on the membrane structure of human red blood cells. Five amino acids tested herein delay the formation of insulin fibril and alter the fibrillar morphology. The alteration of morphology can not afford insulin fibril with different cytotoxicity.