Kubota M
Kanagawa Shigaku. 1989 Jun;24(1):157-68.
The effect of matrix components extracted Bovine periodontal ligament (PDL) on cell proliferation and alkaline phosphatase activity of cultured human periodontal ligament fibroblast (HPLF) were examined in order to understand the cell-tissue interaction of periodontal ligament. Bovine PDL tissue was sequentially extracted with 0.05 M Tris HCl buffer, pH 7.4, containing 1M NaCl or 4M GdmCl. After seeding 24 hours, the cultured HPLF were exposed to the extracts for two through eight days. Nine days after seeding, HPLF indicated four times high activity on ALP and 1.6 times the amount of total protein than those of control (without extract), while DNA synthesis increased only 1.2 times. On the contrary, the NaCl extract depressed the ALP activity of HPLF. The GdmCl extract enhanced both the total protein and ALP activity in dose-dependently. The ALP increasing activity of GdmCl extract on HPLF is stable to heat (78 degrees C, 20 min) and collagenase treatment but partially inactivated by trypsin digestion. Since the GdmCl extract also induced colony formation of NRK-49F cell in soft agarose, it was suggested that the extract contain EGF and TGF-beta like factor. Molecular size of this factor was estimated as 20-50Kd using Sepharose CL-6B gel chromatography. Furthermore, this factor from Sepharose CL-6B were separated into two forms by ion exchange CM-Sepharose column chromatography. Purified preparation from reversed phase column chromatography contained 14Kd, 15Kd, 17Kd, 20Kd, 28Kd40Kd, and 46Kd components on SDS-PAGE. This factor may accumulate in extracellular matrix, and may play a role of cell-tissue interaction and homeostasis in periodontal ligament.
为了解牙周膜的细胞与组织间相互作用，研究了从牛牙周膜（PDL）中提取的基质成分对培养的人牙周膜成纤维细胞（HPLF）增殖及碱性磷酸酶活性的影响。牛PDL组织依次用含1M NaCl或4M GdmCl的0.05M Tris HCl缓冲液（pH 7.4）提取。接种24小时后，将培养的HPLF暴露于提取物中2至8天。接种9天后，HPLF的碱性磷酸酶活性比对照组（无提取物）高4倍，总蛋白量是对照组的1.6倍，而DNA合成仅增加1.2倍。相反，NaCl提取物降低了HPLF的碱性磷酸酶活性。GdmCl提取物剂量依赖性地增强了总蛋白和碱性磷酸酶活性。GdmCl提取物对HPLF的碱性磷酸酶增强活性对热（78℃，20分钟）和胶原酶处理稳定，但经胰蛋白酶消化后部分失活。由于GdmCl提取物还诱导NRK - 49F细胞在软琼脂糖中形成集落，提示该提取物含有表皮生长因子（EGF）和转化生长因子 - β（TGF - β）样因子。使用Sepharose CL - 6B凝胶色谱法估计该因子的分子大小为20 - 50Kd。此外，通过离子交换CM - Sepharose柱色谱法将来自Sepharose CL - 6B的该因子分离为两种形式。反相柱色谱法纯化的制剂在SDS - PAGE上含有14Kd、15Kd、17Kd、20Kd、28Kd、40Kd和46Kd成分。该因子可能积聚在细胞外基质中，并可能在牙周膜的细胞 - 组织相互作用和体内平衡中发挥作用。
