Biochemical Investigation on Amyloid of Ageing in Human Intervertebral Discs.

在滑膜关节中已经报道了人关节结构中与淀粉样蛋白相关的淀粉样蛋白沉积（Uchino等人，1980; Goffin等人，1981）和椎间盘（Takeda等，1984年，通过组织学研究。虽然来自各种组织的淀粉样蛋白的特征在于淀粉样纤维蛋白的性质并根据淀粉样纤维蛋白进行分类，但这种生物化学研究方法尚未应用于关节组织淀粉样蛋白的研究。水的有效性-本研究评价了Pras等人的溶解技术。60例手术椎间盘标本，收集纤维组织和髓核并储存在-20 ℃。根据年龄和保存条件选择30个椎间盘进行生物化学研究，样品在液氮中粉碎后，用150 mM生理盐水提取12次，洗脱大部分可溶性蛋白。通过在280 nm处的吸收监测蒸馏水的连续增溶。收集前两种提取物。经负染后的电镜观察，水溶性部分的电镜观察显示在约100个观察视野内有少量的淀粉样纤维簇。因此，没有尝试进一步的纯化或表征。得出的结论是，椎间盘中淀粉样蛋白的水溶性产生非常少量的淀粉样蛋白纤维，并且组织中淀粉样蛋白含量低或基质的一些未知影响导致提取不良。

Age-associated amyloid deposition in human articular structure has been reported in synovial joints (Uchino et al., 1980; Goffin et al.,1981) and in intervertebral discs (Takeda et al., 1984) by histological studies. While amyloid from various tissues are characterized and classified by the nature of amyloid fibril proteins, such biochemical method of investigation has not been applied to the study of amyloid of articular tissues.To investigate amyloid of ageing in human interverterbral discs biochemically, validity of the water-solubilization technique by Pras et al. was assessed in the present study.Sixty surgical specimen of intervertebral discs consisting of annulus fibrosus and nucleus pulposus were collected and stored at -20 C. Thirty intervertebral discs were selected for biochimical studies by their age and by the conditions after preservation.After pulverization in liquid introgen, tweleve times of extraction of the sample by 150 mM saline washed out most of the soluble proteins. Successive solubilization by distilled water was monitored by absorption at 280 nm. and the first two extracts were collected. Aliquots were processed for electronmicroscopic study after negative staining.Electron microscopic studies of water solubilized fraction showed a few cluster of amyloid fibrils in about 100 observatory fields. Therefore, further purification or characterization was not attempted.It was concluded that water-solubilization of amyloid in intervertebral disc yields very small amount of amyloid fibrils and that a low content of amyloid in the tissue or some nuknown effect of the matrix resulted in poor extraction.