Structural Characters of Cell Attachment and Spreading Factors Deribed from Human Periodontal Ligament Fibroblasts

近年来，为了了解牙周病的发病过程以及牙周组织细胞的功能，细胞外因子和细胞生长因子受到了广泛的关注。细胞的再生可能通过1)细胞的趋化/迁移，2)细胞的附着和铺展，3)细胞的增殖和分化，4)细胞外基质的合成。本研究旨在阐明Kawase T.et al(Adv.Dent.Res.1988)方法培养的人牙周膜成纤维细胞样细胞(HPLF)合成的细胞黏附和扩散因子(CASFs)的特性。将融合的HPLF在添加0.2 mM抗坏血酸-2P的无血清MCDB107培养液中培养24小时。收集HPLF条件培养液(HPLF-CM)，经超滤后，应用于IEC-DEAE柱，在20 mM Tris-HCl pH.8.0中用0~0.5M的氯化钠洗脱。…此外，将HPLF-CM用于0.5M磷酸盐缓冲液pH.7.0洗脱的羟基磷灰石色谱柱。用抗纤维连接蛋白和玻璃体连接蛋白及其受体的抗体对完整和分离的HPLF-CM进行CASF检测。经IEC-DEAE柱层析分离后，用SDS-PAGE对分离得到的CASFS进行纯化。为了证实HPLF合成CASF，用放线菌酮(10ug/ml)孵育的HPLF-CM抑制了CASF的活性。含有RGD细胞附着信号肽的GRGDS肽仅抑制HPLF-CM中约30%的CASF活性，提示CASFS可能有另一个序列可能是受体的结合域。胰酶可切割HPLF-CM的CASF活性，抗体不能改变HPLF-CM的CASF活性，热处理(80ﾟC，15min)可完全抑制HPLF-CM的CASF活性，因此，CASF活性的主要结构域应该是多肽。经DEAE柱层析和SDS-PAGE鉴定，HPLF-CM中含有多个CASF，经0.25M的氯化钠洗脱后，银染的CASF蛋白约为43 KDa。此外，另一个70 KDa的蛋白能与钙化组织如牙骨质的必需成分羟基磷灰石结合，因此推测该蛋白可能是富含酸性氨基酸和/或磷酸化等翻译后修饰的酸性蛋白。较少

Recently, considerable attention has been directed toward extracellular and cell growth factors to understand the processes of periodontal disease as well as the function of the cells periodontium. The cellular events of regeneration might be performed by 1) cell chemotaxis/migration, 2) cell attachment and spreading, 3) cell proliferation and differentiation, and 4) synthesis of extracellular matrix proteins. The purpose of this research projest was to clarify the characterization of cell attachment and spreading factors (CASFs) synthesized by human periodontal ligament derived fibroblast-like cells (HPLF) which were migrated from explantsby the method of Kawase T.et al (Adv.Dent.Res.1988). The confluent HPLF were cultured in the FCS-free MCDB107 medium supplemented with 0.2mM ascorbic acid-2P for 24 hours. The conditioned medium of quiencent HPLF (HPLF-CM) was collected, condenced by a ultrafiltration and applied to IEC-DEAE column eluted with 0 to 0.5M NaCl in 20mM Tris-HCl pH.8.0. … More The HPLF-CM was applied to a hydroxyapatite column eluted with 0.5M phosphate buffer pH.7.0. The CASF assay of intact and fractionated HPLF-CM were performed by the method by Kawase T.et al, using antibodies against fibronectin and vitronectin, and their receptors. The purification of CASFs were demonstrated by SDS-PAGE on fractions which were separated by IEC-DEAE column. In order to confirm that HPLF syntheze the CASFs, HPLF-CM of HPLF incubated with cycloheximide (10ug/ml) inhibited the CASF activity. GRGDS peptide containing RGD cell attachment signal peptide inhibited only approximately 30% of total CASF activity in HPLF-CM.It is suggested that CASFs may have another sequence which could be a binding domain for receptor. Trypsin cleaved the activity of CASF of HPLF-CM.Antibodies didn't alter the CASF activity of HPLF-CM.And heat treatment (80ﾟC,15 min) completely inhibited CASF activity of HPLF-CM.Thus, main domain for CASF activity should be peptide. There were several CASFs in HPLF-CM which were recognized by DEAE column and SDS-PAGE.The CASF eluted with 0.25M NaCl was estimated approximately 43KDa protein stained with a silver staining. And, one of another CASFs which was estimated 70KDa proteinn can bind to hydroxyapatite, which is essential component of calcified tissue such as cementum.Therefore, it was suggested that CASFs could be acidic proteins which may be rich of acidic amino acids and/or posttranslational modification such as phosphorylation. Less