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Development of an alveolar bone resorption inhibitor that suppresses interleukin-1

开发抑制白细胞介素 1 的牙槽骨吸收抑制剂

基本信息

批准号：
02454479
负责人：
KOGA Toshihiko
金额：
$ 4.16万
依托单位：
National Institute of Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

Periodontitis is characterized by alveolar bone loss. Interleukin-1 (IL-1) produced by macrophages and polymorphonuclear leukocytes is considered to play a central role in the bone resorption at inflammatory regions in periodontitis. In this study, we purified an IL-1 inhibitor from culture supernatants of macrophages and examined whether it suppresses the bone resorption.IL-1 inhibitor was purified from culture supernatants of P388D_1 cell line murine macrophages stimulated with lipopolysaccharide for 72 h to homogeneity by a five-step procedure: acetic acid extraction from culture supernatants; Bio-Gel P-60 gel chromatography; DEAE-Sepharose CL-6B column chromatography; reverse-phase high-performance liquid chromatography on a C_<18> hydrophobic support; and high-performance liquid chromatography on a gel filtration column. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 67 kDa on SDS-polyacrylamide gel electrophoresis, and had an isoelectric point of 4.8. The IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. The IL-1 inhibitor suppressed murine and human IL-1-induced proliferation of C3H/HeJ mouse thymocytes. However, the IL-1 inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2 and the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A. The inhibitor at final concentrations of 1 to 5 mug/ml inhibited almost completely in vitro IL-1 induced bone resorption of BALB/c mouse calvaria, suggesting that the inhibitor may be useful for control of bone resorption in periodontitis.

牙周炎的特征是牙槽骨丢失。由巨噬细胞和多形核白细胞产生的白细胞介素-1（IL-1）被认为在牙周炎炎症区域的骨吸收中起着中心作用。本研究从脂多糖刺激小鼠P388D_1细胞72 h的巨噬细胞培养上清中，经乙酸抽提、Bio-Gel P-60凝胶层析、琼脂糖凝胶层析、琼脂糖凝胶层析DEAE-SepharoseCL-6 B柱色谱法、C_2疏水载体反相高效液相色谱<18>法和凝胶过滤柱高效液相色谱法。纯化的IL-1抑制剂在SDS-聚丙烯酰胺凝胶电泳上得到单一条带的蛋白质，分子量为67 kDa，并且具有4.8的等电点。IL-1抑制剂是一种热和酸稳定的蛋白质，其通过用胰蛋白酶消化和用二硫苏糖醇还原而失活。IL-1抑制剂抑制小鼠和人IL-1诱导的C3 H/HeJ小鼠胸腺细胞增殖。然而，IL-1抑制剂并不影响IL-2诱导的IL-2依赖性CTLL-2细胞的增殖和促有丝分裂剂量的刀豆球蛋白A刺激的C3 H/HeJ小鼠胸腺细胞的增殖。终浓度为1 - 5 μ g/ml的抑制剂几乎完全抑制体外IL-1诱导的BALB/c小鼠颅骨骨吸收，表明该抑制剂可用于控制牙周炎中的骨吸收。