AGE-ASSOCIATED CHANGES IN COLLAGEN

这项研究的目的是调查可能的机制。 中观察到的与agp相关的潜在变化 在之前资助的一项研究中，胶原蛋白发生了交联。的确有 有证据表明，有两种机制可以在引入这种 变化：1.现有数据表明，存在一个胶原池 这不是对象的周转；它假设这样的池， 它的大小可能会随着时间的推移而增加，经历渐进的 在动物一生中，分子会发生变化。2.有 有证据表明，在生命后期合成的胶原蛋白可能 与先前合成的胶原蛋白在结构上不同。在这 建议使用体内和体外技术来 调查这些相互交织的问题。为了调查营业额，一个 在大鼠身上进行的体内标记方案将用于测试 有一个胶原池的假设(在操作上 定义为含有分子间、双官能团的胶原 源自异丙基氧化酶生成的醛的交联物)，即 和动物一样老。这样进行的生命周期标签研究 远不能区分不同的胶原蛋白 分子水平上的水池；诸如溶解度等性质 特征通常用于推断结构方面。 在目前的研究中，建议跟踪胶原池 直接通过标记交联物。为了调查 第二种可能的机制，涉及胶原蛋白的差异 由年长的和年轻的动物合成的，包括成纤维细胞和器官 来自不同年龄的老鼠和猴子的培养将被用来 分析胶原蛋白的体外生物合成。费率等参数 生物合成、胶原蛋白类型比率、交联物产生、程度 赖氨酸羟化与酶和非酶 糖基化作用将被测量。老鼠和猴子被选为 短命和长寿物种的例子，如之前的数据 这表明大鼠的胶原蛋白的老化可能与人类的不同。 任何观察到的变化背后可能的机制是 按逻辑顺序进行检查。 已开发出精确、快速的高效液相分析技术。 胶原蛋白的交联物。未分级的组织水解物可以是 在体内标记中的早期时间点直接分析 研究，当标记的交联链的特定活性仍然是 相对较高。在以后的时间点，当特定活动 由于生长和标记的胶原蛋白的存在而下降的组织 制备水解物，通过凝胶过滤进行分析。

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.