The role of glycocylphosphatidylinositol-anchor proteins in cell proliferation and differenciation.

糖酰磷脂酰肌醇锚蛋白在细胞增殖和分化中的作用。

• 批准号: 10672080
• 负责人: NAKABAYASHI Toshikatsu
• 金额: $ 1.66万
• 依托单位: Mukogawa Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672080/
• 关键词: TBR 31-2 cells; proliferation and differentiation; osteoblast; adipocyte; differentiation factor; differentiation marker; GPI-anchor proteins; GPI・アンカー蛋白質; TBR 31・2細胞; GPI-アンカータンパク質

(1) Differentiation capacity of TBR 31-2 cellsTo study the differentiation capacity of TBR 31-2 cells, the m-RNA expression of differentiation factor, osteoblast core binding factor 1and differentiation phenotypes such as alkaline phosphatase, type I collagen, osteocalcin, osteopontin, PTH receptor and vitamin D receptor were examined by reverse transcription-polymerase chain reaction. Expression of these proteins was only observed in the stage of cell differentiation. In addition, the increase of m-RNA expression level in differentiation factor, peroxisome proliferator-activated receptor γ and differentiation markers such as lipoprotein lipase and adipsin were also observed. These observations suggest that TBR 31-2 cells show bipotential characters, differentiation toward adipocytes and osteoblasts.(2) Purification and identification of GPI-anchor proteins in TBR 31-2 cellsAfter biotinylation of peripheral proteins present on the plasma membranes of TBR 31-2 cells, GPI-anchor proteins were released by the treatment of PI-PLC. Solubilized GPI-anchor proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane, and then analyzed chemiluminescence using peroxidase-conjugated streptoavidin. Four proteins having molecular weights of 120, 112, 96 and 60kDa were specifically solubilized by the action of PI-PLC.

(1)TBR 31-2细胞的分化能力为研究TBR 31-2细胞的分化能力，采用逆转录-聚合酶链式反应检测分化因子、成骨细胞核心结合因子1的m-RNA表达及碱性磷酸酶、I型胶原、骨钙素、骨桥蛋白、甲状旁腺素受体和维生素D受体等分化表型。这些蛋白仅在细胞分化期有表达。此外，分化因子、过氧化物酶体增殖物激活受体γ及分化标记物脂蛋白脂酶和脂酶的m-RNA表达水平也升高。这些观察结果表明，TBR 31-2细胞具有向脂肪细胞和成骨细胞分化的双向性。(2)TBR 31-2细胞中GPI锚定蛋白的纯化和鉴定将溶解的GPI锚定蛋白用SDS-PAGE分离，印迹到硝酸纤维素膜上，然后用过氧化物酶标记的链霉亲和素进行化学发光分析。通过PI-PLC的作用，特异性地溶解了四种相对分子质量分别为120、112、96和60 kDa的蛋白质。