Study on the mechanism of terminal differentiation and calcification of cultured mandibular chondrocytes

培养下颌软骨细胞终末分化及钙化机制研究

• 批准号: 04671262
• 负责人: NAKAGAWA Koichi
• 金额: $ 1.34万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671262/
• 关键词: Mandibular Chondrocytes; Type I Collagen; Type II Collagen; Type X Collagen; Alkaline Phosphatase; Osteonectin; Osteopontin; タイプIコラーゲン; タイプIIコラーゲン; タイプXコラーゲン

During the term of the year of 1993, we studied expression of differentiated phenotype of rabbit mandibular chondrocytes in culture. The experiments done yielded the following results.[Microscopic and Histological Analyzes] Rabbit mandibular chondrocytes took flat spindle-like shape resembling fibroblasts in appearance by the day 6 of culture. On the day 10, however, they changed their shape into rounded one characteristic to differentiated chondrocytes. Then the cells produced and secreted out tremendous amount of cartilage-matrix molecules to became hypertrophic by the day 16 of culture. Alcian blue staining became intense by the day 13, and then gradually declined. In contrast, alizarin red staining could be detectable on the day 16, and then increased to become fully positive by the day 27 or later.[Transmission Electron Microscopic Analyzes] While a large amount of type I collagen fiber accumulated in the pericellular space of the cells during the first week of culture, type II collagen fiber was recognized as a major component of interterritorial matrix. The cells after culture for two weeks in vitro appeared to be at the initial calcifying stage. After four weeks, the cells became hypertrophic, but no transdifferentiation into osteoblasts occurred.[Expression of ALPase Activity] The cells expressed a low level of ALPase activity as long as bFGF was present in the culture. When bFGF was withdrawn from the culture at confluence on the day 5 of culture, ALPase activity began to increase. The enzyme activity reached a maximal level on the day 15, and then gradually decreased.[Northern Blot] Expression of type II collagen and ALPase mRNAs reached maximum on the day 7 of culture, and then decreased. Expression of type I collagen mRNA gradually increased time-dependently to become maximal on the day 12. During culture of the cells for four weeks, no expression of type X collagen, osteopontin or osteonectin mRNA was detectable.

1993年，我们研究了兔下颌骨软骨细胞在培养中分化表型的表达。所做的实验产生了以下结果。【镜检及组织学分析】培养第6天，兔下颌软骨细胞呈扁平梭形，外观类似成纤维细胞。然而，在第10天，它们的形状变成了圆形，这是分化软骨细胞的一个特征。然后细胞产生并分泌出大量的软骨基质分子，在培养的第16天变得肥大。到第13天，阿利新蓝染色变得强烈，然后逐渐减弱。相比之下，茜素红染色在第16天可以检测到，然后在第27天或更晚的时候增加到完全阳性。【透射电镜分析】在培养的第一周，细胞的细胞周间隙中积累了大量的I型胶原纤维，而II型胶原纤维被认为是胞间基质的主要成分。体外培养2周后，细胞处于初始钙化阶段。4周后，细胞变肥大，但未发生转分化成成骨细胞。【ALPase活性表达】只要培养中存在bFGF，细胞就能表达低水平的ALPase活性。在培养第5天将bFGF从培养液中撤出，ALPase活性开始升高。酶活性在第15天达到最大值，随后逐渐降低。【Northern Blot】II型胶原蛋白和ALPase mrna的表达在培养第7天达到最大值，然后逐渐降低。I型胶原mRNA的表达随时间逐渐增加，在第12天达到最大值。细胞培养4周后，未检测到X型胶原、骨桥蛋白和骨连接蛋白mRNA的表达。

项目成果

田中栄二: "上顎第二大臼歯の遠心移動を行なった叢生の2治療例について" 矯正臨床ジャーナル. 8. 51-61 (1992)

Eiji Tanaka：“上颌第二磨牙远移治疗拥挤的两个病例”正畸临床杂志，8. 51-61 (1992)。

Y.Duan: "Changes in bone formation during experimental tooth movement after the denervation of rabbit inferior alveolar nerve." J.Osaka Univ.Dent.Sch.33(発表予定). (1994)

Y.Duan：“兔子下牙槽神经去神经后实验性牙齿移动过程中骨形成的变化。”J.Osaka Univ.Dent.Sch.33（待提交）。

M.SAKUDA,K.TANNE,K.YOSHIDA H.INOUE,H.OHMAE,M.TSUCHIYA S.ADACHI,K.NAKAGAWA,Y.INOUE: "Integrated information-processing system in clinical orthodontics:An approach with use of a computer network system." Am.J.Dentfac.Orthop.101. 210-220 (1992)

M.SAKUDA,K.TANNE,K.YOSHIDA H.INOUE,H.OHMAE,M.TSUCHIYA S.ADACHI,K.NAKAGAWA,Y.INOUE：“临床正畸中的集成信息处理系统：一种使用

H.INOUE: "Effects of centrifugal force on growth of mouse osteoblastic MC3T3-E1 cells in vitro." J.Dent.Res.72. 1351-1355 (1993)

H.INOUE：“离心力对小鼠成骨细胞 MC3T3-E1 细胞体外生长的影响。”

MEGUMI: "Differentiated phenotype of mandibular chondrocytes and their transdifferentiation." Jap.Journal of Bone and Mineral Metabolism. 11. 201 (1993)

MEGUMI：“下颌软骨细胞的分化表型及其转分化。”