人类牙乳头间充质细胞向成牙本质细胞定向的诱导分化

Induced differentiation of human dental papilla mesenchymal cells into odontoblasts

目的:探讨人牙乳头间充质细胞向成牙本质细胞的分化机制和分化过程。方法:实验于2004-04/2005-03在四川大学华西口腔医学院口腔生物医学工程教育部重点实验室完成。①实验材料:选择四川大学华西第二医院自然流产的三四个月胎龄的人胚胎(孕妇知情同意,经过医院伦理委员会批准),胰岛素样生长因子Ⅰ和碱性成纤维细胞生长因子(均购自美国Sigma公司)。②实验分组:实验分为空白对照组、胰岛素样生长因子Ⅰ组、碱性成纤维细胞生长因子组和联合组。③实验干预:在建立人牙乳头间充质细胞体外培养模型的基础上,用不含白血病抑制因子的DMEM/F12培养基作为基础培养液,分别用浓度为10μg/L的胰岛素样生长因子Ⅰ、浓度为100μg/L的碱性成纤维细胞生长因子以及同时用这两种因子诱导人牙乳头间充质细胞向成牙本质细胞定向分化。④实验评估:采用倒置显微镜、免疫细胞化学和反转录-聚合酶链反应等方法从形态学和功能方面检测细胞的变化。结果:①培养和诱导细胞形态学变化及碱性磷酸酶活性:诱导细胞培养7d时,联合组和胰岛素样生长因子Ⅰ组的培养细胞均呈现单一胞浆突起,形态上出现成牙本质样细胞改变,细胞的碱性磷酸酶活性明显增高,联合组细胞的胞浆突起更明显,细胞的碱性磷酸酶活性更高;碱性成纤维细胞生长因子组与对照组比较,细胞变化不明显。②细胞免疫化学结果:联合组和胰岛素样生长因子Ⅰ组牙本质涎磷蛋白的免疫细胞化学染色胞浆呈阳性。③细胞hDSPPmRNA的表达:联合组和胰岛素样生长因子Ⅰ组细胞经反转录-聚合酶链反应后获得约599bp的特异性片段,表明两组细胞在mRNA水平表达特异性牙本质涎磷蛋白。而其余两组未见表达。结论:胰岛素样生长因子Ⅰ能刺激人牙乳头间充质细胞向功能性成牙本质细胞样细胞分化,碱性成纤维细胞生长因子可产生协同作用。

AIM： To investigate the differentiation mechanism of human dental papilla mesenchymal cells toward odontoblast lineage. METHODS： The experiment was performed at the Key Laboratory of Oral Biomedical Engineering Ministry of Education of Sichuan University from April 2004 to March 2005. Human dental papilla mesenchymal cells were isolated from human 3 to 4 month old embryos obtained by spontaneous abortion （West China Second Hospital of Sichuan University, informed consent from the pregnant woman, and permission from Hospital Ethics Committee） and cultured in D MEM/F12 culture media without leukemia inhibitory factor. Cells induced with 10 μg/L insulin-like growth factor Ⅰ （IGF-Ⅰ, Sigma） to differentiate toward odontoblast served as IGF-Ⅰ group; induced with 100 μg/L basic fibroblast growth factor （bFGF, Sigma） as bFGF group; with IGF-Ⅰ and bFGF together as the integrated group, and cells without any medicine as the blank control group. The morphological and functional changes of calls were examined by inverted microscope, immunocytochemistry and RT- PCR. RESULTS： ①After 7 days, the cultured cells induced with IGF- Ⅰ were polarized with long processes, and presented odontoblast-like calls, and the alkalinephosphatase activity of cells was remarkably increased; The cells induced with mixed IGF- Ⅰ and bFGF exhibited with longer processes and higher alkaline phosphatase activity compared with IGF- Ⅰ group. There was no obvious changes in cultured cells between IGF- Ⅰ group and control group. ②Immunohistological assay suggested that the immunocytochemistry staining of dentin sialophosphoprotein （DSPP） in kytoplasm was positive in the IGF- Ⅰ and integrated groups. ③The specific segment of 599 bp was obtained in integrated group and IGF- Ⅰ group after RT-PCR, indicating the cells of these two groups expressed hDSPP mRNA by RT-PCR. No expression was found in the other groups. CONCLUSION： IGF- Ⅰ can stimulate human dental papilla mesenchymal cells to differentiate toward odontoblast-like calls, bFGF displays a synergistic inductive effect.