Studies for linage of periodontal ligament cells by gene transfection technique.

为了阐明从牙周韧带（PDL）中分离的细胞谱系，进行了以下研究。首先，我们构建了外源基因导入人PDL细胞原代培养并表达的实验方案。我们构建了下游含有人端粒酶基因的巨细胞病毒即时早期增强子和启动子的质粒载体，并将其引入人细胞外生法建立的原代细胞培养中。用RT-PCR方法证实端粒酶基因在抗生素标记克隆细胞中的表达。其次，分析了碱性磷酸酶（ALP）在人PDL细胞单细胞衍生克隆中的表达，碱性磷酸酶是成骨细胞谱系的经典标记。克隆的细胞形态相同，但ALP表达水平不同，对地塞米松的反应也不同。ALP基因的RNA表达水平在这些克隆中几乎相同，表明ALP活性水平的不同来源于ALP蛋白的翻译和/或跨膜过程。第三，我们研究了人PDL细胞在不同克隆中的基因表达谱。通过微阵列分析两个不同PDL克隆（高ALP克隆和低ALP克隆）中表达的总rna，并在1500多个不同基因中相互比较。这些PDL克隆表现出几乎相同的RNA表达谱，在一些特定基因上略有差异。最后，我们还分析了神经生长因子在小鼠PDL克隆中的作用。

To clarify a lineage of cells isolated from periodontal ligament (PDL), the following studies were undertaken. First, we constructed (he experimental protocol for introducing and expression of the foreign genes into primary culture of human PDL cells. We constructed the plasmid vector which have human telomerase gene at downstream for cytomegarovirus immediate early enhancer and promoter, and introduced it to the primary cell culture which were established by the outgrowth method from human PDL. The expression of telomerase gene in cloned cells selected by an antibiotic marker was confirmed by RT-PCR. Secondly, expression of alkaline phosphatase (ALP) which were known to be a classical marker for cells in osteoblastic lineage were analyzed in single cell derived-clones form human PDL cells. The cloned cells showed same morphology, however expressed different levels of ALP activities and showed different responses against dexamethasone. The RNA expression levels for ALP gene were almost identical among these clones, suggesting that the different levels for ALP activities were derived from translational and/or transmembrane process of ALP protein. Thirdly, we investigated the gene-expression profiles in different clones for human PDL cells. Total RNAs expressed in two different PDL clones, one is high ALP clone and other is low ALP clone, were analyzed by a microarray assay, and compared with each other in over 1,500 different genes. These PDL clones showed almost same RNA expression profiles and showed slight difference in several specific genes. Finally, we also analyzed the roles of nerve growth factors in mouse PDL clones.