smad1/5信号通路在诱导多能干细胞成釉分化中的作用研究

Effects of Smad1/5 Signal in the Ameloblastic Differentiation of Induced Pluripotent Stem Cells

目的 研究smad1/5信号在诱导多能干细胞(induced pluripotent stem,iPS)成釉分化中的作用.方法 采用小鼠胚胎成纤维细胞作为滋养层培养iPS细胞,采用Western blot法检测成釉细胞无血清条件培养液(ameloblast serum-free conditioned medium,ASF-CM)或对照培养基培养iPS细胞14天后,其smad1/5及MAPKs通路蛋白表达情况;分别在ASF-CM中加入smad1/5或MAPKs抑制剂后培养iPS细胞,采用RT-PCR法及免疫荧光染色分别检测iPS细胞成釉标志分子mRNA及阳性细胞率的情况.结果 ASF-CM组iPS细胞p-smad1/5(6.1±1.3)、p-P38 (3.2±0.6)以及p-ERK1/2(4.1 ±0.8)蛋白的表达均较对照组增高(P<0.05),其成釉蛋白(mRNA:6.6±1.3;阳性细胞率:46.6%±11.3%),釉质素(mRNA:5.4±0.9;阳性细胞率:51.4%±10.9%)及细胞角蛋白-14(mRNA:5.9±1.1;阳性细胞率:41.9%±8.1%)mRNA及蛋白的表达较对照组也增高(P<0.05),smad1/5通路抑制剂LDN-193189可显著逆转ASF-CM的上述促iPS细胞成釉分化的效应(P<0.05),但p38MAPK及ERK1/2通路抑制剂对上述效应无显著影响(P>0.05).结论 smad1/5信号通路在ASF-CM诱导的iPS细胞成釉分化过程中发挥关键调控作用.

Objective To investigate the effects of smad1/5 signal in the ameloblastic differentiation of induced pluripotent stem cells(iPS).Methods The mouse embryo fibroblasts were used as the feeder cells for iPS cells.Western blot was used to measure the protein expression of smad1/5 and mitogen-activated protein kinases(MAPKs)by iPS cells cultured with control medium or ASF-CM.In addition,inhibitors of mitogen-activated protein kinases(MAPKs)phosphorylation were used to treat the iPS cells in combination with ASF-CM,and the gene and protein expression of ameloblastin,enamelin and cytokeratin-14 by iPS cells were detected by real-time PCR and immunohistochemical staining.Results ASF-CM treatment significantly increased the protein expression of p-smad1/5(6.1±1.3),p-P38(3.2±0.6)and p-ERK1/2(4.1±0.8)comparing to the controls(P<0.05).In addition,ASF-CM treat-ment significantly increased the mRNA and protein expression of ameloblastin(mRNA:6.6%±1.3%;percentage of positive cell:46.6%±11.3%),enamelin(mRNA:5.4±0.9;percentage of positive cell:51.4%±10.9%)and cytokeratin-14(mRNA:5.9±1.1;percent-age of positive cell:41.9%±8.1%)(P<0.05),which could be obviously reversed by p-smad1/5 inhibitor(P<0.05).Conclusion smad1/5 signal transduction plays an important role during the regulation of the ameloblastic differentiation of iPS cells.