Development of the methods for application of the factors that biologically accelerate reparative dentin formation

1.人离体牙牙髓细胞的分离及表型鉴定在伦理委员会的许可下，从人离体牙中分离牙髓细胞。用RT-PCR方法检测其基因表达谱和表型。结果表明，它们表达成牙本质细胞特异性基因牙本质涎磷蛋白（DSPP），被怀疑是成牙本质细胞谱系.生长因子对它们附着、增殖和分化的影响生长因子，例如，转化生长因子-β 1（TGF-β 1）、碱性成纤维细胞生长因子（bFGF）和结缔组织生长因子（CTGF）对塑料培养皿附着的影响。结果，这些生长因子的吸附增强了它们对塑料培养皿的附着。同时观察了对细胞增殖和碱性磷酸酶（ALP）活性的影响。在增殖方面，只有TGF-β 1促进了它们的增殖。TGF-β 1和bFGF可下调ALP 3的活性。羟基磷灰石（HAP）对它们附着的影响为了评估HAP与髓源性细胞的相容性，研究了它们在HAP上的附着。结果，与由聚苯乙烯制成的塑料皿相比，HAP上的附着显著更高。HAP吸附生长因子有增强附着的趋势，但差异不显著. HAP对其基因表达的影响为了判断HAP对牙髓源细胞基因表达是否有影响，将其接种于HAP上，通过RT-PCR检测其基因表达。结果，DSPP和1型胶原基因表达显著增强。

1. Pulp cell isolation from human extracted tooth and confirmation of their phenotypeUnder permission of ethical committee, pulp cells were isolated from human extracted tooth. RT-PCR was carried our to confirm their gene expression profile and phenotype. As a result, they expressed odont oblast-specific gene, dentin sialophosphoprotein (DSPP), and were suspected to be odont oblast lineage.2. Effects of Growth factors on their attachment, proliferation, and differentiationEffects of growth factors, e.g., transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and connective tissue growth factor (CTGF), on their attachment to plastic dish were investigated. As a result, adsorption of these growth factors enhanced their attachment to plastic dish. Effects on their proliferation and alkaline phosphatase (ALPase) activity were also investigated. Concerning about proliferation, only TGF-beta1 enhanced their proliferation. While ALPase activity was downregulated by TGF-beta1 and bFGF.3. Effects of hydroxyapatite (HAP) on their attachmentTo estimate compatibility of HAP with pulp-derived cells, their attachment onto HAP was investigated. As a result, attachment onto HAP was significantly higher compared to plastic dish made of polystyrene. Adsorption of growth factors onto HAP tended to enhance attachment, but the difference was not significant.4. Effect of HAP on their gene expressionTo estimate that HAP have an effects on gene expression of pulp-derived cells or not, they were seeded onto HAP and their gene expression were investigated by RT-PCR. As a result, DSPP and type 1 collagen gene expression were significantly enhanced.