Investigation for establishment of periodontal regenerative therapy using stem cells in cultured periodontal ligament cells

利用培养牙周膜细胞干细胞建立牙周再生治疗的研究

• 批准号: 20592436
• 负责人: KOBAYASHI Makoto
• 金额: $ 3万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2008
• 资助国家: 日本
• 起止时间: 2008 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20592436/
• 关键词: ヒト歯根膜; 間葉系幹細胞; 歯周組織再生; Side Population(SP)細胞; 骨分化制御; BMPアンタゴニスト; twist; biglycan; decorin; 培養ヒト歯根膜細胞; SP細胞; 組織幹細胞; 腱・靱帯マーカー; ヒト歯根膜細胞; Side Population 細胞; 腱・靭帯マーカー分子

In 2008 : The various types of stem cells exist in side population(SP) effused strongly Hoechst 33342 dye. The SP cells in bone marrow express cell surface markers of human stem cells(HSCs) and are highly enriched for HSC activity. In this study, we clarified the characteristics of SP cells isolated from human periodontal ligament(HPDL) cells. Unfortunately, the present data concluded that HPDL cells contain SP cells, whereas the SP cells are not enriched for mesenchymal stem cell(MSC)-like properties.In 2009, Twist and bone morphogenetic protein(BMP) antagonists may play an important role in maintenance of constancy of PDL, by which negatively regulate osteoblastic differentiation of cells existing in PDL. In this study, changes of these molecules expression by inducing osteoblastic differentiation were assessed in HPDL cells. We further investigated regulatory effects of BMP antagonists on osteoblastic differentiation in the cells. The present results suggest that under physiological … More condition, twist and BMP antagonists ; gremlin and follistatin, which are constitutively expressed in HPL cells, negatively regulate osteoblastic differentiation of the cells. Further, the induction of osteoblastic differentiation in HPL cells simultaneously enhances twist expression, while it suppresses expression of gremlin and follistatin, as the result, the osteoblastic differentiation may be modulated.PDL contains fibroblastic cells, which may participate in formation and maintenance of PDL fibers, and osteoblastic cells with a potential to generate alveolar bone and cementum, however, the specific markers for recognition and isolation of each cell type have not been clarified. The purpose of this study was to isolate alkaline phosphatase(ALP)-positive and-negative subpopulations from HPDL cells using flowcytometry and to identify functional difference between the 2 subsets in terms of mitogenic activity, osteogenic potential and expression of decorin as well as biglycan. The present results suggest that ALP-positive HPDL cells have osteoblast-like phenotype, while ALP-negative HPDL cells are fibroblastic cell population with weak osteogenic potential. Further, biglycan may positively regulate osteoblastic differentiation in ALP-positive HPDL cells, while decorin, whose expression is increased by osteogenic induction, may negatively regulate mineralization in both subpopulations.In 2010, to investigate characteristics of MSC existed in PDL, CD271+/CD90+cells, which were isolated from HPDL by FACS, were single cell cultured, and the clones with MSC-like properties were collected. Therefore, CD271+/CD90+may be a marker to collect MSC from HPDL. Less

2008年：各种类型的干细胞存在于侧群(SP)中，强注入Hoechst 33342染料。骨髓中的SP细胞表达人类干细胞(HSCs)的细胞表面标志，并高度富含HSC活性。在本研究中，我们阐明了从人牙周膜(HPDL)细胞中分离的SP细胞的特性。不幸的是，目前的数据表明，HPDL细胞中含有SP细胞，而SP细胞并不富含间充质干细胞(MSC)样的特性。2009年，Twist和骨形态发生蛋白(BMP)拮抗剂可能在维持PDL的稳定性方面发挥了重要作用，通过对PDL中存在的细胞的成骨分化进行负面调节。在这项研究中，通过诱导成骨细胞分化，评估了这些分子在HPDL细胞中表达的变化。我们进一步研究了BMP拮抗剂对细胞成骨分化的调节作用。目前的结果表明，在生理性…下更多的条件、Twist和BMP拮抗剂；在HPL细胞中结构性表达的gremlin和folistatin，负向调节细胞的成骨分化。此外，在HPL细胞中诱导成骨细胞分化的同时增强了twist的表达，同时抑制了gremlin和folistatin的表达，从而可能调节了成骨细胞的分化。PDL含有可能参与PDL纤维形成和维持的成纤维细胞，以及具有生成牙槽骨和牙骨质的成骨细胞，然而，识别和分离每种细胞类型的特异性标记尚未阐明。本研究的目的是用流式细胞术从HPDL细胞中分离碱性磷酸酶(ALP)阳性和阴性亚群，并从有丝分裂活性、成骨潜能、核心蛋白聚糖和Biglycan的表达等方面鉴定这两个亚群之间的功能差异。提示ALP阳性的HPDL细胞具有成骨细胞样表型，而ALP阴性的HPDL细胞为成纤维细胞群，成骨能力较弱。此外，Biglycan可能正向调节碱性磷酸酶阳性的HPDL细胞的成骨分化，而Decorin在成骨诱导后表达增加，可能负向调节两个亚群的矿化。2010年，为了研究PDL中存在的MSC的特性，用FACS从HPDL中分离出CD271+/CD90+细胞，进行单细胞培养，并收集具有MSC样特性的克隆。因此，CD271+/CD90+可能是从HPDL中采集MSC的标志。较少

项目成果

Characteristics of side cell population cells existing in periodontal ligament cells

牙周膜细胞中存在的侧细胞群细胞的特征

• 发表时间: 2008
• 作者: Koide Y;Kobayashi M;et al.
• 通讯作者: et al.

ヒト歯根膜細胞中に存在するアルカリフォスファターゼ陽性細胞と陰性細胞の特徴

人牙周膜细胞碱性磷酸酶阳性和阴性细胞的特征

• 发表时间: 2009
• 期刊: Dental Med Res 29(1)
• 作者: 鶴見亜有子、小林誠;ら
• 通讯作者: ら

培養ヒト歯根膜細胞におけるTwistと各種BMP antagonistの発現制御

Twist 和各种 BMP 拮抗剂在培养的人牙周膜细胞中表达的调节

• 发表时间: 2008
• 期刊: Dental Med Res
• 作者: 森高俊一郎;小林誠;三井将ら;(2番目)
• 通讯作者: (2番目)

ヒト歯根膜細胞中のアルカリフォスファターゼ陰性細胞の特徴

人牙周膜细胞碱性磷酸酶阴性细胞的特征

• 发表时间: 2008
• 作者: 鶴見亜有子;小林誠;奥津朋彦;ら
• 通讯作者: ら

培養ヒト歯根膜細胞中に存在するSide Population細胞の特徴.

培养的人牙周膜细胞中存在的侧群细胞的特征。

• 发表时间: 2008
• 作者: 小出容子;小林誠;ら
• 通讯作者: ら