Oegema T R, Swedenburg S M, Bradford D S, Thonar E J
Department of Orthopaedic Surgery, University of Minnesota, Minneapolis.
Spine (Phila Pa 1976). 1988 Jun;13(6):707-11.
Chymopapain (1 mg) was injected into each of four-lumbar intervertebral discs of adult mongrel dogs. As expected, at 2 weeks, all injected discs exhibited marked loss of height (mean: 50% of original height) indicative of severe proteoglycan depletion. The appearance of keratan sulfate-bearing fragments in plasma was monitored by an ELISA-inhibition assay which uses a monoclonal antibody (1/20/5-D-4) specific for an epitope present only in the longest keratan sulfate chains. Levels of plasma keratan sulfate rose within 30 minutes and reached a maximum between 24 and 72 hours later. Levels then declined progressively but were still elevated at 2 weeks postinjection. Keratan sulfate-bearing fragments in plasma were purified by ion exchange chromatography on DEAE-Sephacryl and fractionated by sieve chromatography on Sepharose CL-6B. These plasma keratan sulfate-bearing fragments were found to be similar in size to keratan sulfate-bearing fragments generated by chymopapain digestion of dog nucleus pulposus proteoglycans, but slightly larger than single keratan sulfate chains obtained by alkaline borohydride treatment of dog nucleus pulposus proteoglycans. The results of this study show that measurements of blood levels of keratan sulfate could prove useful in monitoring effective degradation of disc proteoglycans in chemonucleolysis in man and help discriminate between ineffective enzyme placement, and alternative mechanisms of treatment failure.
将糜蛋白酶（1毫克）注射到成年杂种狗的四个腰椎椎间盘中。正如预期的那样，在2周时，所有注射的椎间盘都出现了明显的高度损失（平均：原始高度的50%），这表明蛋白聚糖严重耗竭。通过ELISA抑制试验监测血浆中含硫酸角质素片段的出现，该试验使用一种单克隆抗体（1/20/5-D-4），该抗体对仅存在于最长硫酸角质素链中的一个表位具有特异性。血浆硫酸角质素水平在30分钟内升高，并在24至72小时后达到峰值。然后水平逐渐下降，但在注射后2周时仍升高。通过在DEAE-琼脂糖凝胶上进行离子交换色谱法纯化血浆中含硫酸角质素的片段，并通过在琼脂糖凝胶CL-6B上进行分子筛色谱法进行分级分离。发现这些血浆中含硫酸角质素的片段在大小上与糜蛋白酶消化狗髓核蛋白聚糖产生的含硫酸角质素片段相似，但略大于通过碱性硼氢化钠处理狗髓核蛋白聚糖获得的单个硫酸角质素链。这项研究的结果表明，测量血液中硫酸角质素的水平可能有助于监测人体化学髓核溶解术中椎间盘蛋白聚糖的有效降解，并有助于区分无效的酶放置以及治疗失败的其他机制。
