Molecular cloning of the factors that biologically accelerate reparative dentin formation

从生物学角度加速修复性牙本质形成的因子的分子克隆

• 批准号: 12470418
• 负责人: KUBOKI Takuo
• 金额: $ 8.83万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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/en/grant/KAKENHI-PROJECT-12470418/
• 关键词: reparative dentin; gene transfection; adenovirus vector; connective tissue growth factor; transforming growth factor-beta; odontoblast; 炎症; クローニング; アデノウイルスベクター

1. Gene delivery to cultured cellsWe prepared recombinant adenovirus vector carrying beta-galactosidase gene. Mouse osteoblast-like cells, MC-3T3 -E1, were cultured with this vector. As a result, almost all cells were transfected with beta-galactosidase gene.2. Localization of known factors (TGF-beta 1 and CTGF) in vivo animal modelLocalization of TGF-beta 1, that is supposed to be involved in reparative dentinogenesis, and CTGF were confirmed with immunohistochemical staining in animal (wister rat) experimental model. As a result, in 2 weeks from tooth reduction reparative dentin-like tissues were observed, and strong staining of TGF-beta 1 and CTGF were observed around these tissues.3. Gene expression of TGF-beta 1 and CTGF in cultured cells stimulated with proinflammatory factorsMDPC-23, mouse-derived odontoblast-like cells were used in this study. The cells were stimulated with IL-1 beta and bacterial LPS. Changes in CTGF and TGF-b1 genes expression were examined by RT-PCR. As a result, MDPC-23 cells were expressing the CTGF and TGF-beta 1 genes coastitutively, and both factors increased CTGF gene expression and decreased TGF-b1 gene expression in the odontoblast-like cells (MDPC-23) within one-day period after stimulation.4. Effect of TGF-beta 1 and CTGF to cultured cellsThe effects of rCTGF and rTGF-beta 1 on cell proliferation were determined by the MTT assay. rTGF-beta 1 tended to decrease the proliferation dose-dependently, whereas effect of rCTGF was not evident. Next, to investigate the effects of rCTGF on calcification of MDPC-23 cells, cells were cultured with medium containing rCTGF. Sequential addition of AA and b-GP up-regulated the ALPase activity, while addition of rCTGF had no obvious effects.

1.制备了携带β-半乳糖苷酶基因的重组腺病毒载体。用该载体培养小鼠成骨样细胞MC-3 T3-E1。结果，几乎所有细胞都被β-半乳糖苷酶基因转染。2.已知因子（TGF-β 1和CTGF）在体内动物模型中的定位通过免疫组织化学染色在动物（wister大鼠）实验模型中证实了被认为参与修复性牙本质形成的TGF-β 1和CTGF的定位。结果，在离体牙2周内，观察到修复性牙本质样组织，并在这些组织周围观察到TGF-β 1和CTGF的强染色.促炎因子刺激体外培养的成牙本质样细胞中TGF-β 1和CTGF的基因表达本研究采用小鼠源性成牙本质样细胞MDPC-23。用IL-1 β和细菌LPS刺激细胞。RT-PCR检测CTGF和TGF-β 1基因表达的变化。结果显示，MDPC-23细胞呈CTGF和TGF-β 1基因的互补表达，并且在刺激后1天内，两种因子均使成牙本质细胞样细胞（MDPC-23）中CTGF基因的表达增加，而TGF-β 1基因的表达降低. TGF-β_1和CTGF对培养细胞的影响MTT法检测rCTGF和rTGF-β_1对细胞增殖的影响。rTGF-β 1呈剂量依赖性地抑制细胞增殖，而rCTGF的作用不明显。接下来，为了研究rCTGF对MDPC-23细胞钙化的影响，用含有rCTGF的培养基培养细胞。依次加入AA和b-GP可上调ALG活性，而rCTGF对ALG活性无明显影响。

项目成果

W.Sonoyama et al.: "Effects of IL-1beta and LPS on CTGF expression in mouse-derived odontoblast-like cells, MDPC-23"Journal of Bone and Mineral Research. 17. 327 (2002)

W.Sonoyama 等人：“IL-1β 和 LPS 对小鼠成牙本质细胞样细胞 MDPC-23 中 CTGF 表达的影响”《骨与矿物质研究杂志》。

W. Sonoyama et. Al.: "Immunohistochemical Localization of Connective Tissue Growth Factor during Reparative Dentinogenesis, (Special Issue)"Journal of Dental Research 80. 59. (2000)

W.索诺山等人。

T. Mizushima et. Al.: "Effect of CTGF/Hcs24 on Proliferation/Differentiation of Articular Chondrocytes in Culture, (Special Issue IADR Abstracts)"Journal of Dental Research 80. 634. (2000)

T.水岛等。

園山 亘: "修復象牙質形成促進時における結合組織成長因子の局在とその発現制御機構"日本補綴歯科学会雑誌. 45. 135 (2001)

Wataru Sonoyama：“促进修复性牙本质形成过程中结缔组织生长因子的定位及其表达控制机制”日本修复医学会杂志 45. 135 (2001)。

Wataru Sonoyama et al.: "Effects of proinflammatory factors on CTGF expression in odontoblast-like cells"Journal of Dental Research. (in press). (2002)

Wataru Sonoyama 等人：“促炎因子对成牙本质细胞样细胞中 CTGF 表达的影响”牙科研究杂志。