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

牙周膜是一种典型的非钙化结缔组织，位于牙骨质和牙槽骨之间，是一种矿化组织，起着支持牙齿的作用。PL参与牙周组织发育、组织再生和咬合、咀嚼等物理应力的改建过程。取人牙周组织外植体获得人牙周膜成纤维细胞(HPLF)，在D-MEM中添加5%胎牛血清和0.25 mM抗坏血酸-2-P进行传代培养。HPLF与无FCS的MCDB107共同培养24小时。收集HPLF条件培养液(HPLF-CM)，用超滤法浓缩，用阴离子交换层析(IEC-DEAE柱)分离。利用HPLF-CM中包被蛋白质的疏水孔进行细胞黏附和铺展因子分析。SDS-PAGE显示其中一种IEC-DEAE组分含有一种以40 kDa为主要成分的蛋白质。用蛋白质测序系统进行氨基酸序列分析，确定40 kDa的蛋白质为骨连接蛋白(On)(SPARC/BM-40)。牵张后On的表达增加。在胶原凝胶中培养的HPLF通过凝胶收缩显著减少ON的合成。涂覆在疏水孔道上的ON具有CASF活性。用0.28M氯化钠洗脱的部分(IEC-DEAE)含有与辛基-琼脂糖柱结合的CASFS。将1%CHAPS洗脱部分应用于SDS-PAGE，并用抗On单抗进行Western blotting鉴定。On与牙骨质的主要矿物成分羟基磷灰石发生强烈的相互作用。将釉质基质蛋白和HPLF-CM包被于疏水孔壁上，直接与HPLF相互作用，可促进On mRNA HPLF的表达，HPLF产生的On可能是CASF的一种，在牙周再生中起重要作用。

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.