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

基因转染技术研究牙周膜细胞谱系。

• 批准号: 12671997
• 负责人: MIYAMOTO Manabu
• 金额: $ 1.92万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671997/
• 关键词: periodontal ligament; alkaline phosphatase; microarray; dexamethasone; 歯根膜細胞; 骨芽細胞関連遺伝子; RT-PCR; alkaline phosphatase; 遺伝子導入

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.

为了阐明从牙周膜(PDL)分离的细胞的谱系，进行了以下研究。首先，我们建立了将外源基因导入原代培养的人PDL细胞并进行表达的实验方案。我们构建了巨细胞病毒即刻早期增强子和启动子下游含有人端粒酶基因的载体，并将其导入原代细胞培养。用抗生素标记筛选克隆细胞，RT-PCR证实端粒酶基因在克隆细胞中的表达。其次，分析碱性磷酸酶(ALP)在人PDL细胞来源的单细胞克隆中的表达，碱性磷酸酶是成骨细胞系细胞的经典标志。克隆的细胞形态相同，但表达不同水平的ALP活性，对地塞米松的反应也不同。这些克隆中ALP基因的RNA表达水平几乎相同，表明ALP活性的不同水平来源于ALP蛋白的翻译和/或跨膜过程。第三，我们研究了不同克隆的人PDL细胞的基因表达谱。用微阵列技术分析了两个不同的PDL克隆，一个是高ALP克隆，另一个是低ALP克隆，并比较了1500多个不同基因的表达情况。这些PDL克隆显示了几乎相同的RNA表达谱，并在几个特定基因上显示了轻微的差异。最后，我们还分析了神经生长因子在小鼠PDL克隆中的作用。

项目成果

Kamioka, H.: "A three-dimensional distribution of osteocyte processes revealed by the combination of CLS and DIC microscopy"Bone. 28・2. 145-149 (2001)

Kamioka, H.：“结合 CLS 和 DIC 显微镜揭示骨细胞过程的三维分布”Bone 145-149 (2001)。

Kamioka H: "A three-dimensional distribution of osteocyte processes revealed by the combination of CLS and DIC microscopy."Bone. 28-2. 145-159 (2001)

Kamioka H：“结合 CLS 和 DIC 显微镜揭示骨细胞过程的三维分布。”骨。

Miyamoto M: "Cephalometrical and mutational analyses of familial case of Crouzon syndrome"Orthodontic Waves. 60・6. 393-397 (2001)

宫本 M：“克鲁宗综合征家族病例的头影测量和突变分析”Orthodontic Waves 60・6 (2001)。

Tsuboi Y: "Mitogenic effects of neutrophins on a periodontal ligament cell line"J Dent Res. 80・3. 881-886 (2001)

Tsuboi Y：“中性粒细胞对牙周膜细胞系的有丝分裂作用”J Dent Res 80・3（2001）。

Kamioka,H.: "A three-dimensioned distribution of osteocyte processes revealed by the combination of CLS and DIC microscopy"Bone. 28・2(in press). (2001)

Kamioka, H.：“结合 CLS 和 DIC 显微镜揭示骨细胞过程的三维分布”Bone.28·2（出版中）。