Aoki F
Kanagawa Shigaku. 1989 Sep;24(2):311-21.
Saito et al recently reported that the alkaline phosphatase (ALPase) of human periodontal ligament fibroblasts (HPLF) showed remarkably high activity which was similar, but not identical phenotype, to that present in osteoblasts, and suggested that HPLF could be termed as "osteoblastic fibroblast." The present study attempts to explore the ALPase synthesized on HPLF in relation to 1,25(OH)2D3. These HPLF were obtained by the explantation method and then subcultured in D-MEM containing 2 mg FCSP/ml, 50 micrograms ascorbic acid/ml and penicillin/streptomycin after trypsinization. The HPLF were inoculated at a cell density of 1.25 x 10(4) cells/cm2 in culture wells. After 24hr, the HPLF were treated every two days for 7 days with 0.5-10nM 1,25 (OH)2D3. Then, ALPase activity, DNA and protein contents were assayed by the methods using p-nitrophenylphosphate, diaminobenzoic acid and Coomassie Brilliant Blue, respectively. Also, ALPase was prepared from the confluent HPLF incubated with 5 nM 1,25 (OH)2D3 for 12 days, and digested with and without trypsin. The crude ALPase which was solubilized with 10mM Tris-HCl, pH 7.4 containing 0.2 mM MgCl2 and 0.1% NP-40 was applied to 5-15% gradient SDS-PAGE and stained with beta-naphththylacid phosphate and First Blue BB salt in 60 mM borate buffer pH 9.7. The cell growth which was assayed by DNA contents and the incorporation of 3H-thymidine was decreased by 1,25(OH)2D3. On the other hand, ALPase activity was increased approximately 3.6 fold at 6 day by the addition of 5 nM 1,25(OH)2D3. From the separation of ALPase activity on SDS-PAGE, 110 K and 120-130 K ALPase were identified. The 110 K ALPase, which was not changed by 1,25(OH)2D3, was converted to 100K, releasing 10K peptide after trypsin treatment. This 110K ALPase might be tightly associated with cell membrane structure. The 120-130K ALPase was remarkably increased by 1,25(OH)2D3 on SDS-PAGE and completely digested with trypsin. The ALPase in the cultured HPLF might be located not only on the plasma membrane but also in the extracellular matrix. Therefore, 1,25(OH)2D3 may regulate the cell cycle and also the gene expression of ALPase of HPLF.
斋藤等人最近报道，人牙周膜成纤维细胞（HPLF）的碱性磷酸酶（ALPase）表现出显著高的活性，其表型与成骨细胞中的相似但不完全相同，并提出HPLF可被称为“成骨样成纤维细胞”。本研究试图探讨HPLF上合成的ALPase与1,25(OH)2D3的关系。这些HPLF通过组织块培养法获得，然后在胰蛋白酶消化后，接种于含2mg胎牛血清（FCS）/ml、50μg抗坏血酸/ml及青霉素/链霉素的D - MEM中进行传代培养。将HPLF以1.25×10(4)个细胞/cm2的细胞密度接种于培养孔中。24小时后，用0.5 - 10nM的1,25(OH)2D3每两天处理HPLF一次，共处理7天。然后，分别采用对硝基苯磷酸法、二氨基苯甲酸法和考马斯亮蓝法测定ALPase活性、DNA和蛋白质含量。此外，从用5nM 1,25(OH)2D3孵育12天的汇合HPLF中制备ALPase，并分别用胰蛋白酶和不用胰蛋白酶进行消化。用含0.2mM MgCl2和0.1% NP - 40的10mM Tris - HCl（pH 7.4）溶解的粗ALPase应用于5 - 15%梯度SDS - PAGE，并在pH 9.7的60mM硼酸盐缓冲液中用β - 萘基磷酸酯和第一蓝BB盐染色。通过DNA含量和3H - 胸腺嘧啶掺入量测定的细胞生长因1,25(OH)2D3而降低。另一方面，添加5nM 1,25(OH)2D3后，6天时ALPase活性增加约3.6倍。从SDS - PAGE上ALPase活性的分离结果鉴定出110K和120 - 130K的ALPase。110K的ALPase不受1,25(OH)2D3影响，经胰蛋白酶处理后转变为100K，释放出10K的肽段。这种110K的ALPase可能与细胞膜结构紧密相关。120 - 130K的ALPase在SDS - PAGE上因1,25(OH)2D3而显著增加，并被胰蛋白酶完全消化。培养的HPLF中的ALPase可能不仅位于质膜上，还存在于细胞外基质中。因此，1,25(OH)2D3可能调节HPLF的细胞周期以及ALPase的基因表达。
