Studies on inhibitory activity of thrombospondin in biomineralization.

血小板反应蛋白生物矿化抑制活性的研究。

基本信息

批准号：
09671896
负责人：
UENO Akemichi
金额：
$ 1.92万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671896/
关键词：
thrombospondin mineralization extracellular matrix proteins MC3T3-E1 cells antisense oligonucleotides von Kossa staining stable transfectant overexpression Stable transformant 欠損変異株プロテオグリカン MC 3T3-E1細胞 von Kossa 染色 Lac Swich I

项目摘要

TSP1 was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. Bovine TSP1 cDNA containing acomplete ORF of 1170 amino acids has high homologies to its human and mouse counteroarts. Immunohistochemical analyses of bovine anterior teeth with anti-TSP1 antibody, 5G11 revealed that TSP1 was localized at predentin between. Northern blot analysis of total RNA from bovine endodontal and periodontal tissues with bovine TSP1 cDNA showed that two sizes of TSP1 mRNAs were highly expressed in odontoblasts but not in dental pulp and gingiva. To make clear the role of TSP1 in predentin and osteoid, located at interface between mineralized and unmineralized tissues, mouse calvaria-derived osteoblastic cells, MC3T3-E 1 were treated with antisense phosphorothioate oligodeoxynucleotides complementary to TSP1 sequences. The 18mer antisense probe, TAS-2, caused a potent and concentration-dependent increase in the number of mineralized nodules and adecrease in the steady state expression of TSP1 in MC3T3-E1 cells even with submicromolar concentrations in the absence of β-glycerophosphate. The corresponding sense, TS-2 or scrambled oligonucleotide, STAS-2 did not show any effects. Then, MC3T3-E1 cells were transfected with a TSP1 expression vector, pBK-CMV-mTSP1, stably transfected cell lines overexpressing recombinant TSP1 were established, and assayed for TSP1 protein and the capacity to mineralize. Constitutive expression of TSP1 dose-dependently inhibited mineralization by these cells. Furthermore, exogenous purified TSP1 also suppressed the production of a mineralized matrix by MC3T3-E1 cells.These results suggest that TSP1 is a negative regulator of biomineralization by bone and odontoblastic cells and might play a role in protection from mineralization of pulp as well as tissues adjacent to bone.

TSP1 是从参与牙本质形成的牛下颌牙成牙本质细胞中克隆的，含有 1170 个氨基酸的完整 ORF，用抗 TSP1 抗体 5G11 对牛前牙进行免疫组织化学分析，结果显示 TSP1 具有高度同源性。定位于牛牙髓和总 RNA 之间的 Northern 印迹分析。牛 TSP1 cDNA 的牙周组织显示，两种大小的 TSP1 mRNA 在成牙本质细胞中高表达，但在牙髓和牙龈中不表达。衍生的成骨细胞，MC3T3-E 1 用与 TSP1 序列互补的反义硫代磷酸寡脱氧核苷酸处理。 18mer 反义探针 TAS-2 会导致 MC3T3-E1 细胞中矿化结节数量的有效且浓度依赖性增加，并导致 MC3T3-E1 细胞中 TSP1 的稳态表达减少，即使在没有 β-甘油磷酸正义的情况下，即使是亚微摩尔浓度。 TS-2或乱序寡核苷酸、STAS-2没有显示任何效果然后，用TSP1转染MC3T3-E1细胞。建立了表达载体 pBK-CMV-mTSP1，稳定转染的细胞系过表达重组 TSP1，并测定了 TSP1 蛋白和矿化能力。TSP1 的组成型表达剂量依赖性地抑制了这些细胞的矿化。此外，外源纯化的 TSP1 也受到抑制。 MC3T3-E1 细胞产生矿化基质。这些结果表明 TSP1 是骨生物矿化的负调节因子和成牙本质细胞，可能在保护牙髓以及骨附近组织免受矿化方面发挥作用。