两亲性脂肽介导的胰岛素转运及其穿膜机理研究

Insulin is the model drug of protein and peptide drugs. Due to the degradation of proteases and poor permeability of the intestinal membrane, it is ineffective by non-injection administration.Recent results showed that the novel cell-penetrating peptides（C18R6EW）which was synthesized by modifing R8 through Glu and Trp, and then bonded a hydrophobic alkyl chain, had better ability of the penetrating cell membrane than R8. In order to improve the transportation and the stability of insulin, in this project computer molecular simulation methods will be adopted to optimize the system of insulin transportation mediated by C18R6EW. Supramolecular systems of Insulin/R8, Insulin/R6EW and Insulin/C18R6EW are constructed,respectively, and evaluate bioavailability of the system using animal model as a whole. In addition, this project will focus on the mechanism of penetrating cell memebrane and the molecular interaction in the three supramolecular systems. The relation among molecular interaction, bioavailability and mechanism of penetrating cell membrane will be cleared. Finally, the effect of amino acids and C18 chain on the insulin transportation and on the system will be elucidated. The results will not only be in favor of theory and practice of insulin supramolecular system, but also provide new research approaches and methods about the mechanism of penetrating cell membrane.

胰岛素是蛋白质类药物的模型药物，由于蛋白酶降解和细胞膜穿透能力的限制，严重制约了非注射型胰岛素给药的开发和应用。我们近期研究发现，与普通寡聚精氨酸（R8）相比，经谷氨酸、色氨酸修饰并键合一段疏水性烷基链的新型穿膜肽（脂肽，C18R6EW）具有更强的穿膜能力。为了进一步提高其携带胰岛素的转运能力和对酶的稳定性，本项目拟通过计算机分子模拟进一步优化C18R6EW介导的胰岛素转运体系；分别构建R8、R6EW、C18R6EW与胰岛素的超分子体系，结合整体动物模型对其进行生物利用度评价；重点围绕上述超分子体系中胰岛素的跨膜机制和分子间相互作用来明确相关机制；通过研究作用方式、生物利用度以及穿膜机制之间的关系，最终阐明修饰的氨基酸和键合C18链在胰岛素穿膜转运和整个体系中的分子机理。相关研究结果不仅会丰富胰岛素超分子体系的理论和实践，而且会为穿膜肽穿膜机理提供新的理论知识。

已有研究表明，穿膜肽的结构重排和修饰会增强其穿膜能力。经谷氨酸、色氨酸取代寡聚精氨酸（R8）并键合一段疏水性烷基链的新型穿膜肽（脂肽，C18R6EW）具有更强的穿膜能力。并其毒性进行了评价，通过检测细胞的存活量和LDK泄漏的情况，发现三种穿膜肽R8，R6EW和C18R6EW之间没有明显差异，说明都是比较安全的穿膜肽。进一步分别构建并优化了 Insulin/R8、Insulin/R6EW和Insulin/C18R6EW的超分子体系。采用圆二色谱发现超分子前后对胰岛素的二级结构影响很少，这说明超分子化对胰岛素的构象基本没有明显的变化。通过体外蛋白水解酶的稳定性实验，发现Insulin/C18R6EW组具有明显的保护胰岛素降解的作用。超分子体系具有很明显地提高胰岛素的穿透能力。其穿膜能力强弱顺序为：Insulin/C18R6EW>Insulin/R6EW> Insulin/R8>>Insulin。但直到目前为止，穿膜肽的入胞机制仍不完全清楚。我们重点对超分子体系中的胰岛素与穿膜肽的分子间作用以及其穿膜机制进行研究，并发现：胰岛素与穿膜肽之间分子间亲和性：Insulin/C18R6EW>Insulin/R6EW> Insulin/R8。C18R6EW介导的胰岛素内化机制和R6EW、R8不太一样，它既有网格蛋白介导的内吞作用，也有小窝/脂筏介导的内吞作用，是多途径的内吞机制。这阐明了修饰的氨基酸和键合C18 链增加了穿膜肽和胰岛素之间的亲和力，并增加了内吞的途径。该研究结果，丰富了胰岛素超分子体系的理论和实践，而且为穿膜肽穿膜机理提供了新的理论知识。

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