Analysis of osteoclast-activating factor produced by synovial tissues from rheumatoid arthritis mutilans

在全膝关节置换术中，从类风湿性关节炎致残患者的膝关节上获得滑膜组织。细胞经胶原酶分离培养。培养一周后，收集培养上清并浓缩，用Sephadex G-15柱进行凝胶过滤。通过分子量梯度对样品进行分离，加入到在象牙碎片上培养的破骨细胞中，比较样品存在和不存在时破骨细胞的吸收孔形成情况。经样品处理的破骨细胞形成的吸收孔是未经处理的破骨细胞的3倍。将促进吸收孔形成的样品进行电泳，检测所含物质的分子量，检测到约6个0.5 kD ~ 12 kD的峰。如上所述，我们检测了各峰分数对破骨细胞形成吸收孔的影响，与对照组相比，含有分子量约为1 kD的蛋白质的分数显著促进了破骨细胞形成吸收孔。用DE52柱进一步电极性分离该蛋白片段，并将片段加入破骨细胞中确认促进吸收孔形成的活性，得到含有活性蛋白的片段。通过反相高效液相色谱法从该组分中分离出蛋白，将三个峰得到的蛋白分别加入破骨细胞中。来自三个峰之一的蛋白质激活破骨细胞形成吸收孔。我们正在使用气相蛋白质测序仪分析从该峰获得的蛋白质的氨基酸序列，但我们尚未确定蛋白质结构。

The synovial tissue was obtained from the knee joint of patient with rheumatoid arthritis mutilans during total knee replacement. Cells were isolated by collagenase treatment and cultured. After one week of culture, the culture supernatant was collected and concentrated, and subjected to gel filtration using a Sephadex G-15 column. The sample was fractionated by molecular weight gradient and added to osteoclasts cultured on an ivory fragment, and the formation of resorption holes was compared between osteoclasts in the presence and absence of the sample. Osteoclasts treated with the sample formed three-fold more resorption holes than osteoclasts without treatment.The samples that promoted the formation of resorption holes were applied on electrophoresis to examine the molecular weights of contained substances, and about six peaks ranging from 0.5 kD to 12 kD were detected. The effect of each peak fraction on the formation of resorption holes by osteoclasts was examinedas described above, and the fraction containing an approximately 1 kD molecular weight protein significantly promoted the formation of resorption holes by osteoclasts compared with the control.This protein fraction was further fractioneted by electrical polarity using a DE52 column, and the fractions were added to osteoclasts to confirm the activity of promoting resorptionhole formation, and a fraction containing the active protein was obtained was obtained.The protein was isolated from the fraction by reverse-phase high performance liquid chromatography, and each protein obtained from three peaks was added to osteoclasts. Protein from one of three peaks activated the formation of resorption holes by osteoclasts.We are analysing the amino acid sequence of the protein obtained from this peak using a gas phase protein sequencer, but we have not yet determined the protein structure.