人瘢痕疙瘩间充质样干细胞向成骨细胞分化过程中Notch信号表达的研究

Expression of Notch signaling pathway expression in the differentiation of human keloid-derived mesenchymal stem cells into osteoblast

目的研究人瘢痕疙瘩间充质样干细胞向成骨细胞分化过程中,Notch 1~4受体的表达情况,探讨Notch信号通路是否参与调控人瘢痕疙瘩间充质样干细胞的成骨分化。方法经患者知情同意后,分离培养人瘢痕疙瘩间充质样干细胞,采用流式细胞术检测CD29、CD34、CD44、CD45及CD90的表达;免疫细胞化学法检测Oct 4的表达;体外诱导其向成骨细胞分化,诱导3周后行茜素红染色;于诱导前及诱导后2、3周,采用实时定量-多聚酶链反应(RT-PCR)及免疫蛋白印迹法(western blot),分别检测细胞Notch 1~4受体mRNA及蛋白的表达。结果流式细胞术结果表明,分离培养的细胞高表达间充质干细胞表型CD29(98.76%)、cD44(98.11%)、CD90(97.86%),不表达造血干细胞表型CD34(0.05%)、CD45(0.03%);免疫细胞化学法检测多能干细胞标志物Oct4呈阳性表达;向成骨细胞体外诱导3周后,茜素红染色可见明显的钙盐结节。RT-PCR和western blot结果显示,随着诱导时间的延长,Notch 1~4受体mRNA和蛋白的相对表达量逐渐减少,差异有统计学意义(P<0.05)。结论在人瘢痕疙瘩间充质样干细胞向成骨细胞诱导分化过程中,Notch受体信号的表达逐渐减弱,低水平的Notch信号激活可能有利于瘢痕疙瘩间充质样干细胞的成骨分化。

Objective To study the expression of Notch receptor 1 to 4 in the differentiation of human keloid-derived mes- enchymal stem cells （hKMSCs） into osteohlast. To explore whether the Notch signaling pathway participates in the regulation of os- teogenic differentiation of hKMSCs. Methods hKMSCs were isolated and cultured after patients＂ informed consent. The expression of CD29, CD34, CD44, CD45 and CD90 were detected by flow cytometry, and the expression of Oct4 was detected by immunocytochem- istry, hKMSCs were induced to differentiate into the osteoblast in vitro, and alizarin red staining was performed at 3 weeks after induced. RT-PCR and Western Blot were performed to detect the expression of mRNA and protein of Notch receptor 1 to 4 at 2 and 3 weeks be- fore and after induction. Results The results of flow cytometry showed that mesenchymal stem cell phenotype of CD29 （98.76%）, CD44 （96.45%） and CD90 （97.86%） were highly expressed and hematopoietic stem cell phenotype of CD34 （0.05%） and CD45 （0.03%） were expressed at a low level. The results of immunocytochemistry showed that the expression of pluripotent stem cell marker Oct4 was posi- tive. After 3 weeks of osteogenic inductionin vitro, obvious calcium salt nodules could be seen through alizarin red staining. The results of RT-PCR and Western Blot showed that with the prolongation of induction time, the relative expression of mRNA and protein of Notch receptor 1 to 4 was gradually reduced. The difference was statistically significant （P〈0.05）. Conclusion In the differentiation of hKMSCs into osteoblast, the expression of the Notch signaling pathway was gradually weakened. Low level of Notch signal activation was beneficial to the osteogenie differentiationof hKMSCs.