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

类风湿性关节炎滑膜组织产生破骨细胞激活因子的分析

• 批准号: 10671391
• 负责人: TOKUNAGA Hirohiko
• 金额: $ 2.05万
• 依托单位: KANSAI MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671391/
• 关键词: Rheumatoid Arthritis; Osteoclast; Synovial Cell

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.

滑膜组织取自全膝关节置换术中类风湿性关节炎患者的膝关节。通过胶原酶处理分离细胞并培养。培养一周后，收集培养上清液并浓缩，使用Sephadex G-15柱进行凝胶过滤。通过分子量梯度将样品分级，并加入到在象牙碎片上培养的破骨细胞中，并比较存在和不存在样品的破骨细胞之间的再吸收孔的形成。用该样品处理的破骨细胞比未处理的破骨细胞形成多3倍的吸收孔。将促进吸收孔形成的样品用于电泳，以检测所含物质的分子量，检测到约6个峰，范围为0.5 kD至12 kD。如上所述检测各峰级分对破骨细胞形成吸收孔的影响，与对照相比，含有约1 kD分子量蛋白质的级分显著促进破骨细胞形成吸收孔。并将该级分加入破骨细胞中以确认促进再吸收孔形成的活性，获得含有活性蛋白的级分。通过反相高效液相色谱法从该级分中分离蛋白，并将从三个峰获得的每种蛋白质加入破骨细胞中。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。