The pathophysiological role of extracellular matrix proteoglycans in human thyroid diseases.

细胞外基质蛋白聚糖在人类甲状腺疾病中的病理生理作用。

• 批准号: 10671045
• 负责人: EMOTO Naoya
• 金额: $ 2.05万
• 依托单位: Nippon Medical School
• 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-10671045/
• 关键词: heparan sulfate; extracellular matris; FGF; thyroid carcinoma; Graves' disease; proteoglycan

I established a simple and sensitive method for detecting FGF-2-binding heparan sulfate and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied on to a Q-Sepharose anion exchange column. After the column was washed with 10 mM phosphate buffer, one μg of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M NaCl. Heparan sulfate containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profile on Q-Sepharose chromatography, including single peak at 0.7 M NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity wad detected only in the fractions eluting at 0.7 M NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillar thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.

我建立了一种简单而灵敏的方法来检测成纤维细胞生长因子-2结合的硫酸乙酰肝素，并在乳头状甲状腺癌和正常甲状腺中进行了鉴定。甲状腺提取液应用于Q-Sepharose型阴离子交换柱。用10 mM磷酸盐缓冲液洗涤后，将1μg的重组人成纤维细胞生长因子2加入到柱上。色谱柱用氯化钠(0.3-1.5M)梯度洗脱。每一组分都印在硝酸纤维素膜上。硫酸乙酰肝素和成纤维细胞生长因子-2的免疫反应通过膜与特异性抗体的孵育来显示，并通过测量显色产物的密度来定量评估。在正常甲状腺组织中，硫酸乙酰肝素在0.7M和0.9M的氯化钠溶液中呈双峰分布。含有硫酸乙酰肝素的组份也显示出成纤维细胞生长因子-2免疫反应，表明成纤维细胞生长因子-2和硫酸肝素形成了复合体。在乳头状甲状腺癌中，硫酸肝素免疫反应在Q-Sepharose层析上表现出不同的洗脱图谱，包括0.7M氯化钠的单峰和与正常甲状腺相似的单峰。然而，只有在0.7M的氯化钠洗脱组分中才能检测到成纤维细胞生长因子-2免疫反应。人乳头状甲状腺癌中结合成纤维细胞生长因子-2的硫酸乙酰肝素亚群的这种缺失可能导致细胞外基质中可利用的游离成纤维细胞生长因子-2的增加。

项目成果

Onose, H., Emoto N., Dugihara, H., Shimizu, K., and Wakabayashi, I.: "Overexpression of fibroblast growth factor receptor 3 in a human thyroid carcinoma cell line results in overgrowth of the confluent cultures."Eur J Endocrinol. 140. 169-173 (1999)

Onose, H.、Emoto N.、Dugihara, H.、Shimizu, K. 和 Wakabayashi, I.：“人甲状腺癌细胞系中成纤维细胞生长因子受体 3 的过度表达导致汇合培养物过度生长。”Eur

Sugihara, H., Emoto N., Tamura, H., Kamegai, J., Shibasaki, T., Minami, S., and Wakabayashi, I.: "Effect of insulin-like growth factor-I on growth hormone-releasing factor receptor expression in primary rat anterior pituitary cell culture."Neurosci Lett.

Sugihara, H.、Emoto N.、Tamura, H.、Kamegai, J.、Shibasaki, T.、Minami, S. 和 Wakabayashi, I.：“胰岛素样生长因子-I 对生长激素释放的影响

Hiroyuki Onose: "Overexpression of fibroblast growth factor receptor3 in a human thyroid carcinoma cell line resuttsin Overgrowth of the corflusut cultures"European Journal of Endocrinology. 140. 169-173 (1999)

Hiroyuki Onose：“人甲状腺癌细胞系中成纤维细胞生长因子受体 3 的过度表达导致 Corflusut 培养物过度生长”《欧洲内分泌学杂志》。

NAOYA EMOTO: "Fibroblast Growth Factor-2 Free from Extpacellular Matrix is increased in papillary Thyroid Carcinomas and Graves' Thyroid"THYROID. 8. 491-497 (1998)

NAOYA EMOTO：“不含细胞外基质的成纤维细胞生长因子 2 在甲状腺乳头状癌和格雷夫斯甲状腺中增加”。

HITOSHI Sugihara: "Effect of insulin-like growth factor I on growth hormone releasing factor receptor expression in primary rat...."Neuroscience Letters. 276. 87-90 (1999)

HITOSHI Sugihara：“胰岛素样生长因子 I 对原代大鼠生长激素释放因子受体表达的影响......”神经科学快报。