Boonkaew Benjawan, Tompkins Kevin, Manokawinchoke Jeeranan, Pavasant Prasit, Supaphol Pitt
The Petroleum and Petrochemical College, Chulalongkorn University, Soi Chula 12, Phayathai Road, Pathumwan, Bangkok 10330, Thailand.
Biomed Mater. 2014 Apr;9(2):025001. doi: 10.1088/1748-6041/9/2/025001. Epub 2014 Jan 31.
Hyperglycemia in diabetes results in the glycation of long-lived proteins. Protein glycation leads to the formation of advanced glycation end products (AGEs), which are implicated in delayed wound healing and other diabetes-associated pathologies, one of which is periodontal disease. Research into the mechanisms by which glycated long-lived proteins such as collagen exert their effects can allow for the understanding of diabetic pathologies and the development of appropriate treatments. However, the high cost of purified protein can be a limitation for many laboratories around the world. The objective of this study was to develop a low-cost in vitro model of glycated gelatine as an alternative to the glycated collagen model. We investigated the glycation of gelatine type A, a denatured form of collagen, which is low-cost and abundantly available. In this study, gelatine was incubated for 7 days with ribose or methylglyoxal (MG). Cross-linking, autofluorescence and UV-Vis spectrophotometry assays were performed and indicated a dose-dependent linear increase in cross-linking and autofluorescence of gelatine by ribose and MG. MG produced more cross-linking compared to ribose at the same concentrations. The UV-Vis spectra of the glycated gelatines confirmed the presence of AGE fluorophores. Because diabetes is a risk factor for periodontal disease, the effect of the glycated substrates on the basic behaviour of human periodontal ligament (HPDL) cells was evaluated. Glycation dose dependently reduced HPDL attachment and cell spreading, indicating that the novel glycated gelatine substrate affects cell behaviour. These results show that gelatine glycated with ribose or MG can be used as low-cost in vitro models to study the effects of protein glycation on cell behaviour in diabetes and ageing.
糖尿病中的高血糖会导致长寿蛋白的糖基化。蛋白质糖基化会导致晚期糖基化终产物（AGEs）的形成，这些产物与伤口愈合延迟及其他糖尿病相关病理状况有关，其中之一就是牙周疾病。研究糖化的长寿蛋白（如胶原蛋白）发挥作用的机制，有助于理解糖尿病病理状况并开发合适的治疗方法。然而，纯化蛋白的高成本可能会限制全球许多实验室的研究。本研究的目的是开发一种低成本的糖化明胶体外模型，以替代糖化胶原蛋白模型。我们研究了A型明胶（胶原蛋白的变性形式）的糖基化，其成本低且易于获得。在本研究中，将明胶与核糖或甲基乙二醛（MG）孵育7天。进行了交联、自发荧光和紫外可见分光光度法测定，结果表明核糖和MG使明胶的交联和自发荧光呈剂量依赖性线性增加。在相同浓度下，MG比核糖产生更多的交联。糖化明胶的紫外可见光谱证实了AGE荧光团的存在。由于糖尿病是牙周疾病的一个危险因素，因此评估了糖化底物对人牙周膜（HPDL）细胞基本行为的影响。糖基化剂量依赖性地降低了HPDL的附着和细胞铺展，表明新型糖化明胶底物会影响细胞行为。这些结果表明，用核糖或MG糖化的明胶可作为低成本的体外模型，用于研究蛋白质糖基化对糖尿病和衰老过程中细胞行为的影响。
