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目的:探讨b FGF、IGF1、BMP4、TGF-β1联合应用对大鼠牙源性间充质细胞(rat dental mesenchymal cells,r DMCs)成牙本质分化的影响。方法 :应用酶消化法和差速消化法分离、培养大鼠牙源性上皮细胞(r DECs)和r DMCs,经cytokeratin、vimentin免疫荧光染色鉴定其来源。应用茜素红染色、Gomori钙-钴法检测矿化液诱导下r DMCs的矿化能力。应用免疫组织化学染色、图像分析、实时荧光定量PCR和Western印迹法检测在b FGF+IGF1(组1)、TGF-β1+BMP4(组2)、b FGF+IGF1+TGF-β1+BMP4(组3)分别诱导下r DMCs中DSPP、CAP、OPN、OCN的表达差异。采用SPSS14.0软件包对数据进行统计学分析。结果:成功分离培养、鉴定r DECs和r DMCs,经矿化液诱导后的r DMCs钙结节和ALP染色阳性。组1、2中,DSPP、CAP、OPN、OCN m RNA和蛋白的表达水平均显著高于空白对照组4,差异显著(P<0.01),其中组1 CAP、OCN和组2 DSPP、OPN的表达水平最高。结论:r DMCs经矿化诱导后具有成骨特性。b FGF+IGF1显著促进CAP、OCN的表达,加速r DMCs向成牙骨质细胞、成骨细胞分化与牙骨质基质、骨基质的矿化;TGF-β1+BMP4显著促进DSPP、OPN的表达,加速r DMCs向成牙本质细胞、成骨细胞分化与牙本质基质、骨基质的矿化,显示其成骨趋向。4种因子联合应用,并不具备显著的协同作用。
Objective: To investigate the effects of bFGF, IGF1, BMP4 and TGF-β1 on dentin differentiation in rat dental mesenchymal cells (r DMCs). Methods: Rat dental epithelial cells (r DECs) and r DMCs were isolated and cultured by enzymatic digestion and differential digestion. The origin of r DECs and r DMCs were identified by cytokeratin and vimentin immunofluorescence staining. Alizarin red staining and Gomori calcium-cobalt method were used to detect the mineralization ability of r DMCs induced by mineralized solution. Immunohistochemical staining, image analysis, real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of bFGF + IGF1 (group 1), TGF-β1 + BMP4 (group 2), bFGF + IGF1 + TGF-β1 + BMP4 3) induced the expression of DSPP, CAP, OPN and OCN in r DMCs respectively. SPSS14.0 software package for statistical analysis of the data. Results: The r DECs and r DMCs were successfully isolated and cultured, and rMCs calcium positive staining and ALP staining were induced by mineralized solution. The expression levels of DSPP, CAP, OPN, OCN m RNA and protein in group 1 and group 2 were significantly higher than those in control group 4 (P <0.01), and those in group 1 CAP, OCN and group 2 DSPP, OPN The highest level of expression. Conclusion: The r DMCs are osteogenic after being induced by mineralization. b FGF + IGF1 significantly promoted the expression of CAP and OCN, accelerated r DMCs into cementum, osteoblast differentiation and the mineralization of cementum and bone matrix; TGF-β1 + BMP4 significantly promoted the expression of DSPP and OPN , Accelerate r DMCs to odontoblasts, osteoblastic differentiation and mineralization of dentin matrix, bone matrix, showing its tendency to osteogenic. Four kinds of factors combined application, does not have significant synergies.