梯度弹性模量凝胶诱导牙髓干细胞向成牙本质细胞分化的研究

摘要

目的:探讨梯度弹性模量固体凝胶诱导牙髓干细胞(DPSCs)向成牙本质细胞分化的特点.方法:原代分离培养和鉴定小鼠DPSCs;利用低熔点琼脂糖与改良EaSe培养基分别配制成弹性模量＜25、25 ～ 50、50 ～75、75 ～ 100 kPa 4个梯度的固体凝胶,其所对应的琼脂糖浓度分别为4.8、19.2、76.8、307.2 g/L.将第4代DPSCs分别接种于上述凝胶表面,21 d后用茜素红染色检测其矿化结节的形成情况,RT-PCR和Western Blot检测其牙本质基质蛋白-1(DMPl)、涎蛋白(DSP)、磷蛋白(DPP)的mRNA和蛋白表达水平.结果:流式细胞术鉴定结果表明,所培养细胞高表达间充质干细胞标记蛋白CD29和CD90,几乎不表达造血干细胞标记蛋白CD34和CD45;DPSCs诱导分化结果显示,随着弹性模量增高,固体凝胶表面形成的矿化结节随之增多,同时成牙本质细胞标记基因DMPl、DSP、DPP的mRNA和蛋白质表达水平也逐级增加.结论:DPSCs具有顺培养基质表面硬度梯度逐级向成牙本质细胞分化的特点.%AIM:To study the differentiation of dental pulp stem cells (DPSCs) into odontoblasts induced by gel with gradient elastic modulus.METHODS:Mouse DPSCs of passage 4 were seeded on the gel surface with 4 gradient elastic modulus respectively:less than 25 kPa,25 ～ 50 kPa,50 ～ 75 kPa and 75 ～ 100 kPa,which were prepared by a mixture of low melting point agarose and modified Eagle medium,and cultoured for 21 d.The mineralized nodules in the culture were detected using alizarin red staining,and the mRNA and protein expression of dentin matrix protein 1 (DMP1),dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) was tested by reverse transcription polymerase chain reaction and western blot method respectively.RESULTS:The cultured cells expressed CD29 and CD90,almost did not express CD34 and CD45.With the increase of elastic modulus of the solid gel,the mineralized nodule in the culture increased,and mRNA and protein expression level of DMP1,DSP and DPP also increased.CONCLUSION:DPSCs may differentiate into odontoblasts when seeded on the gel surface with different elastic modulus in a gradient dependent form.