Ruel Nancy, Markova Dessislava Z, Adams Sherrill L, Scanzello Carla, Cs-Szabo Gabriella, Gerard David, Shi Peng, Anderson D Greg, Zack Marc, An Howard S, Chen Di, Zhang Yejia
*Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL †Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, PA ‡Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA §Department of Medicine, Section of Rheumatology, Perelman School of Medicine, and Philadelphia Veterans Affairs Medical Center, University of Pennsylvania, Philadelphia, PA ¶Department of Biochemistry, Rush University Medical Center, Chicago, IL ‖Philadelphia Veterans Affairs Medical Center, and Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; and **Dow Agrosciences, Indianapolis, IN.
Spine (Phila Pa 1976). 2014 Jul 15;39(16):1274-9. doi: 10.1097/BRS.0000000000000397.
The presence of fibronectin fragments (FN-fs) and the cleaving enzyme, A disintegrin and metalloproteinase domain-containing protein (ADAM)-8 were examined in human intervertebral disc (IVD) tissue in vitro.
To investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue.
The 29-kDa FN-f has been shown to result in extracellular matrix loss in rabbit IVDs. However, the concentration of this biologically active fragment in the degenerative human IVD tissue has previously not been determined. Furthermore, it is critical to identify the enzyme(s) responsible for FN cleavage in the IVD.
Human degenerative IVD tissues were removed during spinal surgery. A normal seeming young adult and an infant human cadaveric sample were obtained as controls. Soluble proteins were extracted, and analyzed by Western blotting using antibodies specific for the human FN neoepitope VRAA²⁷¹. A purified 29-kDa FN-f was used to allow estimation of the concentration of FN-fs in the tissues. ADAM-8, a FN-cleaving enzyme, was analyzed by Western blotting and immunostaining.
All adult IVD tissues contain many FN-f species, but these species were absent from the infant disc tissue. Moderately degenerative discs contained the highest amount of FN-fs; the concentration was estimated to be in the nanomolar range per gram of tissue. ADAM-8, known to cleave FN resulting in the VRAA²⁷¹ neoepitope, was present in the human disc. ADAM-8 primarily localized in the pericellular matrix of the nucleus pulposus tissue, as determined by immunostaining.
This is the first report that N-terminal FN-fs are consistently present in IVD tissues from adult subjects. The pathophysiological concentration of these fragments is estimated to be at nanomolar range per gram of IVD tissue. Furthermore, ADAM-8, known to cleave FN, is present at the pericellular matrix of disc cells.
在体外对人椎间盘（IVD）组织中的纤连蛋白片段（FN-fs）和裂解酶——含解整合素和金属蛋白酶结构域蛋白（ADAM）-8进行检测。
研究人IVD组织中FN-fs及其裂解酶ADAM-8的存在情况和病理生理浓度。
已证实29-kDa FN-f会导致兔IVD细胞外基质丢失。然而，此前尚未测定这种生物活性片段在退变人IVD组织中的浓度。此外，确定IVD中负责FN裂解的酶至关重要。
在脊柱手术过程中获取人退变IVD组织。获取看似正常的年轻成人和婴儿尸体样本作为对照。提取可溶性蛋白质，并使用针对人FN新表位VRAA²⁷¹的抗体通过蛋白质印迹法进行分析。使用纯化的29-kDa FN-f来估算组织中FN-fs的浓度。通过蛋白质印迹法和免疫染色分析FN裂解酶ADAM-8。
所有成人IVD组织均含有多种FN-f，但婴儿椎间盘组织中不存在这些种类。中度退变的椎间盘所含FN-fs量最高；估计每克组织中的浓度处于纳摩尔范围。已知可裂解FN产生VRAA²⁷¹新表位的ADAM-8存在于人椎间盘中。通过免疫染色确定，ADAM-8主要定位于髓核组织的细胞周基质中。
这是首份关于N端FN-fs始终存在于成人IVD组织中的报告。这些片段的病理生理浓度估计为每克IVD组织处于纳摩尔范围。此外，已知可裂解FN的ADAM-8存在于椎间盘细胞的细胞周基质中。
