权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Dynamic Aspect of Mechanical Stretching on the Synthesis of Cell Attachment and Spreading Factors of Human Periodontium Derived Cells.

机械拉伸对人牙周源性细胞细胞附着和扩散因子合成的动态影响。

基本信息

批准号：
11672091
负责人：
KAWASE Toshio
金额：
$ 2.3万
依托单位：
Kanagawa Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The periodontal ligament (PL) is a typical noncalcified connective tissue that functions on tooth support and lies between cementum and alveolar bone which are mineralized tissues. PL is involved in the remodeling processes with the development of periodontium, tissue regeneration and physical stresses such as occlusion and mastication. Human periodontal ligament derived fibroblasts (HPLF), which were obtained by the explants of human periodontal tissue, were subcultured in D-MEM supplemented with 5% FCS and 0.25mM ascorbic acid-2-P.At the confluent condition. HPLF were cultured with FCS-free MCDB107 for 24 hrs. The conditioned medium of HPLF (HPLF-CM) was collected, condensed by an ultrafiltration method and fractionated with an anion exchange chromatography (IEC-DEAE column). The cell attachment and spreading factor assay was performed by using a hydrophobic well coated with proteins in HPLF-CM.SDS-PAGE revealed that one of the fraction of IEC-DEAE contained a protein with molecular weight of 40kDa as a major component. Amino acid sequence analysis using a protein sequencing system identified 40kDa protein as osteonectin (ON) (SPARC/BM-40). The expression of ON was increased by stretching. HPLF cultured in a collagen gel dramatically decreased the synthesis of ON by gel contraction. ON coated on a hydrophobic well has CASF activity. The fraction eluted with 0.28M NaCl (IEC-DEAE) contained CASFs which binded to Octyl-Sepharose column. 1%CHAPS eluted fraction was applied to SDS-PAGE and identified by the Western blotting method using monoclonal anti-ON antibody. ON was strongly interacted with hydroxyapatite which is major mineral component of cementum. The expression of ON mRNA HPLF was increased by the enamel matrix protein and HPLF-CM which were coated on a hydrophobic well and directly interacted with HPLF.ON produced by HPLF could be one of CASF and play an important role of in periodontal regeneration.

牙周韧带（PL）是一种典型的非钙化结缔组织，在牙齿支撑上发挥作用，位于牙骨质和牙槽骨（矿化组织）之间。 PL 参与牙周组织发育、组织再生和咬合和咀嚼等物理压力的重塑过程。将人牙周组织外植体获得的人牙周膜衍生成纤维细胞（HPLF）在补充有5％FCS和0.25mM抗坏血酸-2-P的D-MEM中传代培养至汇合条件。 HPLF与不含FCS的MCDB107一起培养24小时。收集HPLF的条件培养基(HPLF-CM)，通过超滤法浓缩，并通过阴离子交换色谱(IEC-DEAE柱)分级。使用 HPLF-CM 中涂有蛋白质的疏水孔进行细胞附着和扩散因子测定。SDS-PAGE 显示 IEC-DEAE 的一个级分含有分子量为 40kDa 的蛋白质作为主要成分。使用蛋白质测序系统进行氨基酸序列分析，将 40kDa 蛋白质鉴定为骨连接素 (ON) (SPARC/BM-40)。 ON的表达通过拉伸而增加。在胶原凝胶中培养的 HPLF 通过凝胶收缩显着减少 ON 的合成。涂在疏水孔上的 ON 具有 CASF 活性。用 0.28M NaCl (IEC-DEAE) 洗脱的级分含有与辛基琼脂糖柱结合的 CASF。将1%CHAPS洗脱级分应用于SDS-PAGE，并使用单克隆抗ON抗体通过Western印迹法进行鉴定。 ON与羟基磷灰石有强烈的相互作用，羟基磷灰石是牙骨质的主要矿物成分。釉质基质蛋白和HPLF-CM包被在疏水孔上并直接与HPLF相互作用，增加ON mRNA HPLF的表达。HPLF产生的ON可能是CASF之一，在牙周再生中发挥重要作用。