虎杖苷诱导人脐带间充质干细胞向神经元样细胞分化过程中Notch信号通路的表达改变

Effects of Polydatin Combined with Growth Factors on Neuro-differentiation of Haman Umbilical Cord Mesenchymal Stem Cells and Expression of Notch Signal Molecules

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 effects of polydatin combined with cytokine on inducing Haman umbilical cord mesenchymal stem cells（hUCMSCs） to differentiate neuron- like cells. Methods： The hUCMSCs were isolated and expanded in culture. The passage 4 of hUCMSCs was used and induced by polydatin and growth factors. The cellular morphology was observed under microscope, lmmunofluorescence was employed to identify the surface antigens of Class III beta tubulin（ β-tubulin-Ⅲ ）. The qRT- PCR was used to detect the mRNA expression levels of β-tubulin-Ⅲ ,microtubule -associated protein 2 （MAP2）, orphan nuclear receptor correlation factor 1 （Nurrl）, Notchl and hairy and enhancer of split 1 （ HES1 ）. Results ： The hUCMSCs became shrinkage with high refraction and extended distinct processes around cell, displayed neuron - like morphology after induction. The mRNA expression level of β-tubulin-Ⅲ mRNA was significantly higher in low - dose polydatin ＋ growth factors group and high - dose polydatin ＋ growth factors group than that in control and growth factors groups（ P 〈 0.05 ） , and lowest in control（ P 〈 0.05 ）. The expression of MAP2 mRNA was significantly up regulated after induction（ P 〈 0.05 ）, and greatest in high -dose polydatin ＋ growth factors group （P 〈 0. 05 ）. The expressions of Nurrl, Notchl and HES1 mRNA were significantly up regulated in high -dose polydatin ＋ growth factors group than those in other three groups. And the results from immunofluorescence showed that more posi-tive β-tubulin-Ⅲ proteins were found in high - dose polydatin ＋ growth factors group and low - dose polydatin ＋ growth factors group than those in growth factors group, hardly positive β-tubulin-Ⅲ cells were determined in control. Conclusion ： The hUCMSCs can be induced into neuron - like cells successfully by polydatin which was dose - dependent, and the Notch signal pass - way was suppressed. The low level of Notch signaling activation may contribute to neural cell differentiation.