论文部分内容阅读
目的:探讨虎杖苷联合生长因子诱导人脐带间充质干细胞(hUCMSC)向神经元样细胞分化的过程及Notch信号分子的表达。方法:体外分离培养hUCMSC。取第4代细胞,在不同剂量的虎杖苷和生长因子诱导下向神经样细胞分化。诱导后显微镜下观察细胞形态变化,应用细胞免疫荧光技术检测神经元特异性标志物3型β微管蛋白(β-tubulin-Ⅲ)的蛋白表达,荧光定量PCR法检测β-tubulin-Ⅲ、微管相关蛋白2(MAP2)、孤束核受体相关因子1(Nurr1)及Notch通路中Notch1受体蛋白和的靶基因发状分裂相关增强子1(hairy and enhancer of split 1,HES1)表达。结果:诱导后部分细胞胞体收缩变圆,折光性增强,突起变细增多,末端出现分支,呈典型的神经元样细胞。各诱导组的β-tubulin-Ⅲ、MAP2基因表达均高于对照组。β-tubulin-ⅢmRNA在低剂量和高剂量虎杖苷联合生长因子组高于单纯生长因子诱导组,MAP2 mRNA的表达在高剂量虎杖苷联合生长因子组最高。Nurr1 mRNA仅在高剂量虎杖苷联合生长因子组显著上调。细胞免疫荧光结果显示,低剂量和高剂量虎杖苷联合生长因子组部分细胞β-tubulin-Ⅲ表达阳性,单纯生长因子诱导组仅少量细胞表达β-tubulin-Ⅲ,对照组几乎不表达,与mRNA变化一致。Notch1和HES1的mRNA在高剂量虎杖苷联合生长因子组表达下调。结论:虎杖苷能够诱导hUCMSCs分化为神经元样细胞,并抑制Notch信号分子表达,且存在剂量依赖性。低水平的Notch信号激活可能有利于神经细胞的分化。
Objective: To investigate the process of cellard glycoside combined with growth factor inducing human umbilical cord mesenchymal stem cells (hUCMSCs) to differentiate into neuron-like cells and the expression of Notch signaling molecules. Methods: hUCMSCs were isolated and cultured in vitro. The 4th generation cells were taken and differentiated into neuron-like cells under the induction of different doses of polydatin and growth factor. The morphological changes of the cells were observed under a microscope. The protein expression of neuron specific marker β-tubulin-Ⅲ was detected by immunofluorescence technique. The expression of β-tubulin-Ⅲ was detected by fluorescence quantitative PCR. (MAP2), Nurr1, Notch1 receptor protein and target gene hairy and enhancer of split 1 (HES1) in the Notch pathway. Results: After induction, the somatic cells became contracted and rounded, the refraction was enhanced, and the protrusions became thinner and more distal to the branches, showing typical neuron-like cells. The expressions of β-tubulin-Ⅲ and MAP2 in each induction group were higher than those in the control group. β-tubulin-Ⅲ mRNA was higher in low-dose and high-dose groups of polydatin combined with growth factors than in simple-growth-induced group. The expression of MAP2 mRNA was the highest in high dose of polydatin combined with growth factors. Nurr1 mRNA was upregulated only in the high-dose of polydatin combined with growth factors. The results of immunofluorescence showed that β-tubulin-Ⅲ was positive in some cells of low-dose and high-dose groups of PDGF combined with growth factors, but β-tubulin-Ⅲ was only expressed in small amount of cells in simple growth-inducing group, but not in control group Change consistent. The mRNAs of Notch1 and HES1 were down-regulated in the high dose of PDX combined with growth factors. Conclusion: Polydatin can induce hUCMSCs to differentiate into neuron-like cells and inhibit the expression of Notch signaling molecules in a dose-dependent manner. Low levels of Notch signaling may contribute to neural cell differentiation.