Effect of mechanical stress on osteogenesis facilitating by parathyroid hormone

机械应力对甲状旁腺激素促进成骨的影响

• 批准号: 15591974
• 负责人: ARAKAWA Toshiya
• 金额: $ 2.24万
• 依托单位: Health Sciences University of Hokkaido
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591974/
• 关键词: mechanical stress; parathyroid hormon; osteoblast; PTH受容体; 重力負荷; 骨細胞

Parathyroid hormone (PTH) is a hormone that facilitate both osteogenesis and osteoclastic bone resorption. Osteogenesis was stimulated by intermittent administration of PTH, and bone resorption was atimulted repeated administration. However, its mechanism is unclear. We recently found that fluid shear stress, one of mechanical stress induced expression of mRNA of PTH receptor in MC3T3-Y4-A2 cells established from osteocytes in mouse long bone. In order to clarify the effects of PTH on osteogenesis, we constructed fusion protein with PTH receptor and Green fluorescence protein (GFP). First, we amplified a full length of cDNA of PTH receptor from cDNA that was synthesized from mRNA of MC3T3-Y4-A2 cells. Then, the full length of cDNA of PTH receptor was ligated into pEGFP-N1 and -C3 vectors (clontech co.). The EGFP vectors with PTH receptor cDNA were transfected into MC3T3-E1 oseoblast like cells established from mouse bone, and the cells stably expressing PTH receptor/GFP fusion protein were selected by neomysin. However, the cells that had high signals of fluorescence were not isolated because the cells that had bright signals were died. Though the reason was unclear, it assumed that excess signals of cAMP were transmitted in cells. The cells that had resistance to neomysin were finally isolated though the green fluorescence were not observed. The expression of mRNA of the fusion protein were identified by RT-PCR. We are examining the effects of mechanical stress on expression of PTH receptor with GFP.

甲状旁腺激素(PTH)是一种促进成骨和破骨细胞性骨吸收的激素。间歇给予甲状旁腺素刺激成骨，多次重复给予骨吸收。然而，其机制尚不清楚。我们新近发现，机械应力之一的流体剪应力诱导了小鼠长骨成骨细胞建立的MC3T3-Y4-A2细胞中PTH受体的mRNA表达。为了阐明甲状旁腺素在成骨中的作用，我们构建了甲状旁腺素受体与绿色荧光蛋白(GFP)的融合蛋白。首先，我们从MC3T3-Y4-A2细胞的mRNA中扩增出PTH受体的全长cDNA。然后，将甲状旁腺素受体的全长cDNA连接到pEGFP-N1和-C3载体(Clontech Co.)。将携带甲状旁腺素受体基因的绿色荧光蛋白载体导入小鼠成骨细胞系MC3T3-E1中，用新霉菌素筛选出稳定表达甲状旁腺素受体/绿色荧光蛋白融合蛋白的细胞。然而，有高荧光信号的细胞并没有被隔离，因为有明亮信号的细胞已经死亡。虽然原因尚不清楚，但它假设cAMP的过量信号是在细胞内传递的。虽然没有观察到绿色荧光，但最终分离到了对新霉菌素有抗性的细胞。RT-PCR检测融合蛋白的mRNA表达。我们正在用绿色荧光蛋白检测机械应力对甲状旁腺素受体表达的影响。

项目成果

株化骨細胞MLO-Y4-A2におけるPTH受容体遺伝子のメカニカルストレスによる発現

机械应力诱导骨细胞系MLO-Y4-A2中PTH受体基因的表达

• 发表时间: 2003
• 期刊: 東日本歯学雑誌 23
• 作者: 岡山三紀;荒川俊哉;谷村明彦;溝口到;田隈泰信
• 通讯作者: 田隈泰信

Mechanism of PTH receptor induction by fluid shear Stress in the osteocyte-like cell line MLO-Y4-A2

骨细胞样细胞系MLO-Y4-A2中流体剪切应力诱导PTH受体的机制

• 发表时间: 2004
• 期刊: HIGASHI NIPPON DENTAL JOURNAL VOL.23,NO 1
• 作者: Miki Okayama;Toshiya Arakawa;Akihiko Tanimura;Itaru Mizoguchi;Taishin Takuma
• 通讯作者: Taishin Takuma

Abiko Y, et al.: "Upregulated expression of human beta defensin-1 and -3 mRNA during differentiation of keratinocyte immortalized cell lines, HaCaT and PHK16-0b"J Dermatol Sci. 31(3). 225-228 (2003)

Abiko Y 等人：“角质形成细胞永生化细胞系 HaCaT 和 PHK16-0b 分化过程中人 β 防御素 -1 和 -3 mRNA 的表达上调”J Dermatol Sci。