Shuckett R, Malemud C J
Mech Ageing Dev. 1986 Mar;34(1):73-90. doi: 10.1016/0047-6374(86)90106-5.
Six cases of non-pathological articular cartilage were studied by organ explant culture to assess alterations in tissue sulphated proteoglycans (PGs) as a function of time in culture and donor age. Neosynthesized, 35SO4-labeled and endogenous, or already existing, uronic acid-containing PG populations were studied at several time points over 3-4 weeks. PG extractability did not vary with donor age. The proportion of non-extractable endogenous, but not of neosynthesized, PGs increased with time in culture. Sepharose CL-2B chromatography of neosynthesized and endogenous PGs eluted with associate buffer (0.5 M sodium acetate, pH 5.8) revealed 4 PG subpopulations with Kavs of 0.05, 0.28, 0.68 and 0.9-1.0. With culture time, the percentage distribution of newly synthesized PG subpopulations of large hydrodynamic size increased significantly with a concomitant decrease in the relative amount of smaller PGs. Isopycnic cesium chloride density gradients were performed on pooled Sepharose CL-2B peaks under associative (0.5 M GuHCl) and dissociative (4 M GuHCl) conditions to assess component subclasses of PG aggregates and PG monomers within each PG subpopulation. An analysis of the Kav, 0.05 subpopulation indicated an enrichment in dense PG aggregate (A1) and PG monomer (D1). Both A1 and D1 decreased with in vitro age parallelled by an increase in the respective subclasses of least buoyant density, A4 and D4. Sepharose CL-2B chromatography of D1 fractions within this PG subpopulation indicated a progressive decrease in PG monomer hydrodynamic size with time in culture. In contrast to these age-in-culture related alterations in neosynthesized PGs, the endogenous PGs showed neither a significant change in distribution of PG subpopulations nor PG subclasses over the time period of study. These findings showed the ability of human articular cartilage to alter the profile of neosynthesized PG while maintaining the in situ PG population during in vitro cartilage aging. Such findings suggest that this system may be useful in the elucidation of specific changes in articular cartilage PGs associated with time in culture.
通过器官外植体培养研究了6例非病理性关节软骨，以评估组织硫酸化蛋白聚糖（PGs）随培养时间和供体年龄的变化。在3 - 4周的多个时间点研究了新合成的、35SO4标记的以及内源性的（即已存在的）含糖醛酸的PG群体。PG的可提取性不随供体年龄而变化。不可提取的内源性PG（而非新合成的PG）的比例随培养时间增加。用结合缓冲液（0.5M醋酸钠，pH 5.8）对新合成的和内源性的PG进行琼脂糖CL - 2B层析，显示出4个PG亚群，其洗脱体积（Kav）分别为0.05、0.28、0.68和0.9 - 1.0。随着培养时间的延长，流体力学尺寸大的新合成PG亚群的百分比分布显著增加，同时较小PG的相对量减少。在结合（0.5M盐酸胍）和解离（4M盐酸胍）条件下，对合并的琼脂糖CL - 2B峰进行等密度氯化铯密度梯度离心，以评估每个PG亚群内PG聚集体和PG单体的组成亚类。对Kav为0.05的亚群分析表明，致密PG聚集体（A1）和PG单体（D1）富集。随着体外培养时间的延长，A1和D1均减少，同时浮力密度最小的相应亚类A4和D4增加。对该PG亚群内D1组分进行琼脂糖CL - 2B层析表明，PG单体的流体力学尺寸随培养时间逐渐减小。与新合成PG中这些与培养年龄相关的变化相反，在研究时间段内，内源性PG的PG亚群分布和PG亚类均未出现显著变化。这些发现表明，人关节软骨在体外软骨老化过程中能够改变新合成PG的特征，同时维持原位PG群体。这些发现表明，该系统可能有助于阐明与培养时间相关的关节软骨PG的特定变化。
