AGE-ASSOCIATED CHANGES IN COLLAGEN

本研究的目的是探讨可能的机制 已经观察到的与银相关的潜在变化， 胶原交联在以前资助的研究。 有 有证据表明，两种机制可能是有效的， 1.现有的数据表明，存在一个胶原蛋白池， 这不是受试者周转率;据推测， 随着时间的推移，它的大小可能会增加， 在动物一生中的分子变化。 2.有 有证据表明，在生命后期合成的胶原蛋白可能是 与之前合成的胶原蛋白结构不同。 在这 建议体内和体外技术都将用于 调查这些相互交织的问题。 为了调查营业额， 在大鼠中进行的体内标记方案将用于测试 假设存在胶原蛋白池（操作上 定义为含有分子间、双功能 衍生自异戊酰氧化酶产生的醛的交联）， 和动物一样老。 因此进行了寿命标签研究 至今还不能区分不同胶原蛋白 在分子水平上的池;诸如溶解度 特征通常用于推断结构方面。 在本研究中，建议跟踪胶原库 直接通过标记交联。 探讨 第二种可能的机制，涉及胶原蛋白的差异 由年老和年幼的动物合成，包括成纤维细胞和器官 来自不同年龄的大鼠和猴子的培养物将用于 体外分析胶原合成。 速率等参数 生物合成，胶原类型比例，交联生产，程度 赖氨酸羟基化，酶促和非酶促 将测量糖基化。 选择大鼠和猴作为 短寿命和长寿命物种的例子，如以前的数据 这表明胶原蛋白在大鼠中的老化可能不同于在人类中的老化。 任何观察到的变化背后的可能机制将是 按逻辑顺序检查。 精确和快速的HPLC技术已经被开发用于分析 胶原蛋白交联。 未分级的组织水解产物可以是 在体内标记的较早时间点直接分析 研究，当标记的交联的比活性仍然 相对较高。 在稍后的时间点，当特定活动 由于生长和存在标记的胶原蛋白、组织 制备水解产物用于凝胶过滤分析。

The purpose of this study is to investigate possible mechanisms underlying ag associated changes that have been observed in collagen crosslinking in a previously funded study. There is evidence that two mechanisms may be operative in introducing such changes: 1. Available data suggest that a pool of collagen, exists that is not subject turnover; it is hypothesized that such a pool, which may increase in size over time, undergoes progressive molecular changes over the lifetime of the animal. 2. There is evidence that collagen synthesized later in life may be structurally different from collagen synthesized earlier. In this proposal both in vivo and in vitro techniques will be used to investigate these intertwined issues. To investigate turnover, an in vivo labelling protocol conducted in rats will be used to test the hypothesis that there is a pool of collagen (operationally defined as that collagen containing intermolecular, difunctional crosslinks derived from Isyyl oxidase-generated aldehydes) that is as old as the animal. Lifetime labelling studies performed thus far have not sen able to distinguish between different collagen pools at the molecular level; such properties as solubility characteristics are generally used to infer structural aspects. In the present study it is proposed to track collagen pools directly through labelling of crosslinks. To investigate the second possible mechanism, concerning differences in collagen synthesized by older and younger animals, both fibroblast and organ cultures from rats and monkeys of different ages will be used to analyze collagen biosynthesis in vitro. Such parameters as rate of biosynthesis, collagen type ratios, crosslink production, extent of lysine hydroxylation and enzymatic and nonenzymatic glycosylation will be measured. Rats and monkeys were selected as examples of a short lived and long-lived species, as previous data suggest that collagen may age differently in rats than in humans. Possible mechanisms underlying any observed changes will be examined in logical sequence. Precise and rapid HPLC techniques have been developed for analysis of collagen crosslinks. Unfractionated tissue hydrolysates may be analyzed directly at earlier time points in the in vivo labelling study, when specific activity of the labelled crosslinks is still relatively high. At later time points, when specific activity drops due to growth and presence of labelled collagen, tissue hydrolysates are prepared for analysis by gel filtration.