A new diagnostic method for periodontal diseases by determining hepatocyte growth factor in gingival cervical fluid with a high sensitive ELISA.

高灵敏ELISA检测牙龈宫颈液中肝细胞生长因子的牙周病诊断新方法。

• 批准号: 08557100
• 负责人: DAIKUHARA Yasushi
• 金额: $ 8.7万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557100/
• 关键词: Hepatocyte growth factor; Scatter factor; Periodontal disease; Diagnosis; Gingival cervical fluid; High sensitive ELISA; Tissue erpair; HGF; 歯肉溝滲出液; 歯周炎; SF; ELISA; 高感度化

The degree of periodontitis may reflect the concentration of hepatocyte growth factor (HGF) in gingival cervical fluid (GCF) because HGF is thought to be involved in regeneration of various tissues. However, sample volume of GCF is too little (several microliter) to determine the concentrations of HGP by current ELISA for HGF used in clinics (the sensitivity is 200 pg/ml sample). Therefore, we developed two other high sensitive sandwich ELISA systems for HGP as follows. 1. Using mouse anti-HGF monoclonal antibody as the first antibody and chicken anti-HGF IgY conjugated with biotin as the second antibody, we obtained the sensitivity of 20 pg/ml. 2. Using mouse anti-HGF monoclonal antibody as the first antibody and beta-galactosidase conjugated Fab' of rabbit anti-HGF lgG as the second antibody, we obtained the sensitivity of 2 pg/ml. Using the latter ELISA, we found that concentrations of HGF in GCF collected by periopaper from patients with periodontitis were 1-6 ng/ml, and that the concentrations were positively correlated with the depth of periodontal pocket (probing depth) and the gingival index. The concentrations of HGF in GCF in healthy controls were less than 1 ng/ml. Because the average concentration of HGF in serum of healthy subjects is 0.2 ng/ml, these findings suggest that HGP is synthesized and secreted in the inflamed gingival tissues and is involved in regeneration of the tissue. Our finding also suggest the high sensitive ELISA for HGF we developed in this project may be useful for diagnosis of periodontal diseases.

牙周炎的严重程度可能反映了龈沟液中肝细胞生长因子（HGF）的浓度，因为HGF被认为参与各种组织的再生。然而，GCF的样品体积太小（几微升），无法通过目前临床上使用的用于HGF的ELISA（灵敏度为200 pg/ml样品）来测定HGP的浓度。因此，我们开发了另外两种用于HGP的高灵敏度夹心ELISA系统如下。1.以鼠抗HGF单克隆抗体为第一抗体，鸡抗HGF IgY与生物素偶联为第二抗体，获得了20 pg/ml的灵敏度。2.以鼠抗HGF单克隆抗体为第一抗体，兔抗HGF IgG的Fab'-β-半乳糖苷酶偶联物为第二抗体，获得了2 pg/ml的灵敏度。用后一种ELISA方法检测牙周炎患者龈沟液中HGF的浓度为1-6 ng/ml，且浓度与牙周袋深度（探诊深度）和牙龈指数呈正相关。健康对照组龈沟液中HGF浓度均小于1 ng/ml。由于健康受试者血清中HGF的平均浓度为0.2ng/ml，这些发现表明HGP在发炎的牙龈组织中合成和分泌，并参与组织的再生。我们的研究结果也表明，我们在本项目中开发的高灵敏度的ELISA检测HGF可能是有用的牙周疾病的诊断。

项目成果

Harvey,P.: "Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology." Br.J.Cancer. 77. 1052-1059 (1998)

Harvey,P.：“间皮瘤细胞系中 HGF/SF 的表达及其对细胞运动、增殖和形态的影响。”

大西,智和: "HGFの構造と機能" 肝胆膵. 38 印刷中. (1999)

Onishi，Tomokazu：“HGF 的结构和功能”肝胆胰 38 出版中（1999 年）。

Miyata,K.: "Levels of human hepatocyte growth factor (hHGF) in peritoncal fluid after partial hepatectomy." Hepato-Gastroenterol.43. 1594-1600 (1996)

Miyata,K.：“部分肝切除术后腹膜液中人肝细胞生长因子 (hHGF) 的水平。”

Daikuhara, Y.: "Patent pending "Antibodies to hepatocyte groawth factor and the assay method of hepatocyte growth factor using the antibodies"" Patent pending #9-248202. (1997)

Daikuhara, Y.：“正在申请专利“肝细胞生长因子的抗体以及使用该抗体的肝细胞生长因子的测定方法””正在申请专利

Arakaki,N.: "Involvement of oxidative stress in tumor cytotoxic activity of hepatocyte growth factor/scatter factor." J.Biol.Chem.274(in press). (1999)

Arakaki,N.：“氧化应激参与肝细胞生长因子/分散因子的肿瘤细胞毒活性。”