A novel method for histidinohydroxylysinonorleucine measurement and its clinical aaplication

组氨酸羟赖氨酸正亮氨酸测定新方法及其临床应用

• 批准号: 08670946
• 负责人: ISHIKAWA Osamu
• 金额: $ 0.83万
• 依托单位: Gunma University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670946/
• 关键词: Histidinohydroxylsinonorleucine; HHL; Systemic sclerosis; Hypoxia; Low seru concentration; Three dimensional culture; Lysyl oxidase; 全身性強皮症

Histidinohydroxylysinonorleucine (HHL) is a trivalent type of cross-link in skin, which provides collagen for stability and other physiological properties. We established a novel highly sensitive method to measure HHL by using reversed phase HPLC after labeling skin hydrolysates with 9-fluorenylmethyl chrolformate. We measured the content of HHL in the skin specimens of patients with systemic sclerosis and compared it with the HHL of age- and site-matched control skin specimens using this method. Half of the skin sample was processed for a conventional histological examination and chose skin samples that showed the characteristic histological change of SSc, thickened and hyalinized collagens in the entire dermis. The content of HHL in the SSc was significantly increased compared with that of controls when expressed as the molecular ratio of HHL to collagen. The increased amount of HHL in collagen molecules may be associated with the pathological fibrosis of the disease. It is worth inv … More estigating if this increase is specific for SSc or not, or which facto (s) regulates the formation of intermolecular cross-links in vivo.We investigated the effects of hypoxia and low serum concentartion on the expression of extracellular matrix-related genes and the formation of trifunctional intermolecular cross-link of type I collagen by dermal fibroblasts. In the studies of reverse transcriptase polymerase chain reaction (RT-PCR), both hypoxia and low serum concentration revealed the increased mRNA expression of transforming growth factor-beta and the decreased mRNA expression of metalloproteinase-1 in the monolayr culture of dermal fibroblasts from normal individuals and patients with systemic sclerosis. In the three-dimensional culture supplemented with ascorbic acid 2-phosphate, hypoxia remarkably suppressed the formation of histidinohydroxylysinonorleucine, a mature type of trifunctional intermolecular cross-link of type I collagen.The altered mRNA expression of transforming growth factor-beta and metalloproteinase- 1 may well account for the pathoetiology of the post inflammatory stage of systemic sclerosis. The suppressed formation of histidinohydroxylysinonorleucine under the culture condition of hypoxia or low serum concentartion disagreed with the previous in vivo finding. Further studies are required to elucidate factor (s) to regulate the formation of trifunctional intermolecular cross-link of type I collagen. Less

组氨酸是皮肤中的一种三价交联物，它为皮肤提供稳定的胶原蛋白和其他生理特性。建立了用9-荧甲酸甲酯标记皮肤水解物后，用反相高效液相色谱法测定HHL的新的高灵敏度方法。我们用这种方法检测了系统性硬化症患者皮肤标本中HHL的含量，并与年龄和部位匹配的对照皮肤标本进行了比较。对一半的皮肤样本进行常规组织学检查，并选择表现出SSC的组织学特征的皮肤样本，整个真皮中的胶原层增厚和透明。以HHL与胶原的分子比表示，SSC中HHL的含量明显高于对照组。胶原分子中HHL含量的增加可能与疾病的病理纤维化有关。这是值得的inv…为了进一步研究这种增加是否是SSc所特有的，或者是哪个事实(S)调节了活体分子间交联物的形成。我们研究了低氧和低血清浓度对真皮成纤维细胞细胞外基质相关基因表达和I型胶原三功能分子间交联物形成的影响。在逆转录聚合酶链式反应(RT-PCR)的研究中，低氧和低血清浓度均显示正常人和系统性硬化症患者真皮成纤维细胞单层培养中转化生长因子-β基因表达增加，金属蛋白酶-1基因表达降低。在添加抗坏血酸2-磷酸的三维培养中，缺氧显著抑制I型胶原成熟的三功能分子间交联型组氨酸羟赖氨酸的形成，转化生长因子-β和金属蛋白酶-1基因表达的改变可能是系统性硬化症炎症后阶段的发病机制之一。在低氧或低血清浓度培养条件下抑制组氨酸的形成与以往体内发现的结果不一致。调节I型胶原三功能分子间交联物形成的因子(S)尚需进一步研究。较少

项目成果

Sato M,Ishikawa O,Miyachi Y: "Distinct expression of collagen genes by normal and sacr fibroblasts in the the novel three dimensional culture." Br J Drmarol. (in press).

Sato M、Ishikawa O、Miyachi Y：“在新型三维培养物中，正常成纤维细胞和骶成纤维细胞对胶原蛋白基因的不同表达。”

Kondo.A, Ishikawa.O, Okada.K, Miyachi.Y, Abe.S, Kuboki.Y: "Measurement of histidinohydroxylysinonorleucine and hydroxyproline in skin collagen by reversed phase HPLC after 9-fluorenylmethylchrolformate labeling." Anal Biochem. 252. 255-259 (1997)

Kondo.A、Ishikawa.O、Okada.K、Miyachi.Y、Abe.S、Kuboki.Y：“9-芴基甲基氯甲酸酯标记后，通过反相 HPLC 测量皮肤胶原蛋白中的组氨酸羟赖氨酸正亮氨酸和羟脯氨酸。”

Ishikawa.O, Kubota.Y, Miyachi.Y: "Prostaglandine E,suppresses the production of TNF-α and IL-10 by penpheral bloodmononuclear cells." Eur J Pharmacol. (in press). (1998)

Ishikawa.O、Kubota.Y、Miyachi.Y：“前列腺素 E 抑制脑外血单核细胞产生 TNF-α 和 IL-10。”（出版中）。

Ishikawa O, et al.: "Mature type of collagen cross-link,histidinohydroxylysinonorleucine,is significantly increased in the skin of systemic sclerosis." Arthritis Rheum. 41. 376-377 (1998)

Ishikawa O等人：“成熟型胶原蛋白交联，组氨酸羟赖氨酸正亮氨酸，在系统性硬化症的皮肤中显着增加。”

Sato M, et al.: "Opposite effects of tumor necrosis factor-α on type I and type III collagen-gene expression by human dermal fibroblasts in monolayer and three dimensional culture." Br J Dermatol. 138. 118-121 (1998)

Sato M 等人：“肿瘤坏死因子-α 对单层和三维培养中人真皮成纤维细胞的 I 型和 III 型胶原蛋白基因表达的相反影响。” Br J Dermatol。