BMP-2蛋白通过Smad途径对牙髓干细胞成牙本质分化的作用机制研究

The Mechanism of BMP-2 Protein on Odontoblast Differentiation of Dental Pulp Stem Cells Via Smad Pathway

目的:探讨骨形成蛋白2(Bone morphogenetic protein-2,BMP-2)对牙髓干细胞成牙本质分化的影响及对Smad1/5通路的调节作用。方法:取对数生长期的人牙髓干细胞,配制成密度为3×10^(4)个/毫升的细胞悬液,接种于24孔板,置于培养箱中培养至细胞密度约为80%时,将细胞随机分为对照组、单纯诱导组和抑制剂组。对照组细胞不做任何处理,单纯诱导组细胞用浓度为100 ng/ml的BMP-2诱导液(BMP-2溶于DMEM培养基)培养,抑制剂组细胞先用200 nmol/L的Smad1/5抑制剂LDN-193189处理24 h后,更换为BMP-2诱导液,培养14 d后,CCK-8检测人牙髓干细胞活性、qRT-PCR检测牙本质基质蛋白1(DMP-1)和牙本质涎磷蛋白(DSPP)mRNA相对表达量、检测碱性磷酸酶(ALP)活性、茜素红染色观察矿化结节情况、蛋白印迹法检测BMP-2和p-Smad1/5蛋白相对表达量。结果:单纯诱导组矿化结节明显,抑制剂组矿化结节减少;与对照组比较,单纯诱导组牙髓干细胞活性、ALP活性增强,DMP-1和DSPP mRNA相对表达量、BMP-2和p-Smad1/5蛋白相对表达量升高(P<0.05);与单纯诱导组比较,抑制剂组牙髓干细胞活性、ALP活性减弱,DMP-1和DSPP mRNA相对表达量、BMP-2和p-Smad1/5蛋白相对表达量降低(P<0.05)。结论:BMP-2可诱导人牙髓干细胞成牙本质分化,可能是通过调节Smad1/5信号通路发挥作用。

Objective To investigate the effect of bone morphogenetic protein 2(BMP-2) on odontoblast differentiation of pulp stem cells and its regulation on Smad1/5 pathway. Methods The human dental pulp stem cells in logarithmic growth stage were taken and prepared into cell suspension with density of 3×10^(4) cells/ml, then cell suspension were inoculated in a 24-well plate and cultured in an incubator until the cell density was about 80%, then were randomly divided into control group, simple induction group and inhibitor group, control group without any treatment, while simple induction group were treated with 100 ng/ml of BMP-2 and inhibitor group were treated with 200 nmol/L of Smad1/5 inhibitor LDN-193189 for 24 h. Then BMP-2 induction solution was replaced and cultured for 14 days. CCK-8 was used to detect the activity of human pulp stem cells, qRT-PCR was used to detect the mRNA relative expression levels of dentin matrix protein 1(DMP-1) and dentin sialophosphoprotein(DSPP), alkaline phosphatase(ALP) activity was detected, alizarin red staining was used to detect the mineralized nodules, and western blot was used to detect the relative protein expression of bone morphogenetic protein(BMP-2) and p-Smad1/5. Results Mineralization nodules were increased in the simple induction group, while decreased in the inhibitor group. Compared with the control group, pulp stem cell activity, ALP activity, mRNA relative expression levels of DMP-1 and DSPP, protein relative expression levels of BMP-2 and p-Smad1/5 were increased in the single induction group(P<0.05). Compared with the simple induction group, the pulp stem cell activity, ALP activity, mRNA relative expression levels of DMP-1 and DSPP, protein relative expression levels of BMP-2 and p-Smad1/5 were decreased in the inhibitor group(P<0.05). Conclusion BMP-2 can induce odontoblast differentiation of human pulp stem cells, possibly by regulating the Smad1/5 signaling pathway.