基于血管糖萼芯片模型的糖萼损伤和修复机制研究

Diabetic vascular complication is the leading cause of death in patients with the disease, and one of the earliest symptoms occurred in this disease is the damage of vascular glycocalyx caused by high blood sugar. Maintaining the structural and functional integrity of the vascular glycocalyx is a reasonable solution for the prevention and cure of diabetic vascular complications. However, the importance of the vascular glycocalyx haven’t been recognized yet. Because of the particularity of its structure, research of vascular glycocalyx is limited by the traditional ways of cell culture and animal study, resulting in the unclearance of the mechanism of glycocalyx damage and recovery. Microfluidic chip, due to its unique characteristics of dynamic culture and fine control, is believed as the ideal platform to establish in vitro vascular glycocalyx model. In order to study the dynamic change of glycocalyx in diabetes, our group built up tentative models of glycocalyx physiology and damagement by high glucose based on microfluidic chip. On the basis of the prelimanary research, this project aims to improve the established physiologic and damage vascular glycocalyx model, and build up the recovery model further. Based on the vascualr glycocalyx on chip platform, the action mechanism of vascular glycocalyx damage and recovery will be explored tentatively. Finally, the study on chip will be validated with animal model studies. The fullfiiment of this project will help to clarify the action mechanisms of vascular glycocalyx damage and recovery preliminarily. It will also confirm the importance of vascular glycocalyx in diabetic vascular complications, and provide certain references for the prevention and cure of the disease.

糖尿病血管并发症是其患者最主要的致死原因，该病发生的最早期症状之一是高血糖导致的血管糖萼损伤。维护血管糖萼结构和功能的完整性是糖尿病血管并发症防治的潜在措施，而目前普遍对其认识不足。糖萼因其结构特殊性，被传统细胞培养方式和动物实验所局限，其损伤和修复机制迄今不明。微流控芯片具有独特的流动培养和精细控制特性，为血管糖萼研究提供了理想平台。为了研究血管糖萼在糖尿病条件下的动态变化和机制，本课题组初步建立了血管糖萼芯片的生理和高糖损伤模型。本项目拟在前期研究基础上，进一步完善糖萼生理和损伤模型，并建立糖萼修复模型，进而开展对糖萼损伤和修复机理的探讨，最后应用动物试验模型对糖萼芯片模型和机制研究进行验证。本项目研究有望初步阐释血管糖萼高糖损伤和修复的分子机制，进一步明确血管糖萼在糖尿病并发症中的重要作用，为其防治提供一定参考。

血管糖萼损伤是糖尿病等血管疾病发生的最早期症状之一，维护血管糖萼结构和功能的完整性是糖尿病血管并发症防治的潜在措施。关于血管糖萼在糖尿病中的动态变化和机制目前尚未报道。.血管糖萼因其结构特殊性，需要在体外建立更符合体内状态的培养模型。进行了基于微流控芯片的血管糖萼生理、高糖损伤和SDX（舒洛地特药物）修复模型的构建：基于血管形态和血液流动形式，设计制作了多条直通道并排的芯片，便于操作；在芯片上种下细胞，施加一定流速，使之产生10-23 dyn/cm2的剪切力，与体内动脉血流剪切力相当。通过荧光显微镜和共聚焦荧光显微镜3D重构技术在内皮细胞表面看到了明显的糖萼层，经测量其厚度为微米级别，与体内状态相似，建立了糖萼生理模型；在糖萼生理模型中添加葡萄糖，使终浓度为25 mM，与糖尿病人血糖浓度相当。荧光显微镜观察糖萼，通过与对照对比，计算糖萼的损伤率达到30.1%，糖萼高糖损伤模型建立；在糖萼损伤模型中添加200 ug/mL SDX，荧光显微镜观察糖萼，与空白对照对比，计算糖萼的修复率达到114%，糖萼高糖损伤修复模型建立。.基于上述三种模型，研究了高糖以及高糖+SDX下糖萼整体及其组成成分透明质酸（HA）和硫酸软骨素（CS）的变化。发现糖萼整体以及HA和CS均不同程度受到高糖影响而发生损伤，加入SDX后可以不同程度地修复糖萼；研究了高糖对血管内皮细胞的活力、氧化还原状态以及NO产生能力。发现高糖不影响细胞活力，但是可以增加胞内的氧化应激水平，降低NO合成能力。而SDX可以缓解这种情况。.为了进一步研究高糖对HA的损伤机制，通过RT-PCR测定了上述三种模型中HA合成酶（HAS1-3）/降解酶（HYAL1-4，SPAM1）mRNA的含量变化。发现高糖会同时上调HA合成酶和降解酶的表达，但是降解酶的种类和变化幅度大于合成酶。而SDX可以缓解这种情况。进一步通过通过转录组学对三种模型中的细胞信号通路进行探索，发现了8000多条差异基因。.本项目系统研究了血管糖萼各组分在糖尿病下的受损状态及加药后的修复情况，初步阐释了血管糖萼高糖损伤和修复的分子机制，这对基于血管糖萼靶标防治糖尿病等血管疾病提供一定参考。