Yoshihara Y, Plaas A, Osborn B, Margulis A, Nelson F, Stewart M, Rugg M S, Milner C M, Day A J, Nemoto K, Sandy J D
Department of Biochemistry, Rush University Medical Center, Chicago, IL, USA.
Osteoarthritis Cartilage. 2008 Nov;16(11):1343-55. doi: 10.1016/j.joca.2008.04.004. Epub 2008 Jun 3.
We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids.
DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR.
Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth.
The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.
我们研究了炎症相关的α-胰蛋白酶抑制剂（IαI）成分、比库宁、重链（HC）1和HC2在正常软骨、骨关节炎（OA）软骨及滑液中的存在情况。
设计/方法：采用针对比库宁、HC1和HC2的多种抗体，通过蛋白质印迹法研究正常供体和晚期OA患者的软骨提取物以及OA患者的滑液。通过免疫组织化学确定细胞和基质定位，通过逆转录聚合酶链反应（RT-PCR）确定信使核糖核酸（mRNA）。
比库宁-硫酸软骨素（CS）和IαI在OA软骨中含量丰富，但在正常软骨中几乎检测不到。在OA软骨和正常软骨中，HCs主要以一种新的C末端截短的50 kDa形式存在，其中大部分（如果不是全部）与比库宁以外的蛋白聚糖上的CS相连。OA患者的滑液中含有比库宁-CS和与透明质酸（HA）相连的全长（约90 kDa）HCs，即HC-HA（SHAP-HA）。免疫组织化学显示比库宁和HCs的细胞内及细胞相关染色，这与其由表层软骨细胞合成一致。对多个人类正常和OA软骨样本进行的PCR检测到HC1和HC2的转录本，但未检测到比库宁的转录本。在OA软骨中，免疫染色主要与基质相关，在有血管翳样纤维增生的区域最为强烈。
HCs的截短结构、它们与比库宁以外的蛋白聚糖的连接、PCR数据以及细胞内染色均与人类关节软骨细胞合成HC1和HC2一致。OA软骨中存在比库宁-CS和IαI，而正常软骨中不存在，这似乎是由于通过纤维化（但未深度裂开）的软骨表面扩散吸收和保留所致。
