红景天苷通过Wnt/β-catenin信号通路影响小鼠骨髓间充质干细胞向神经元细胞定向分化

摘要

目的:以小鼠骨髓间充质干细胞系D1细胞为研究对象,探讨Wnt/β-catenin信号通路介导的红景天苷诱导D1细胞向神经细胞的定向分化.方法:实验分为对照组(D/F12 完全培养基)和红景天苷诱导组(100 μg/mL+ D/F12 完全培养基).将细胞分别诱导12、24、48和72 h后,采用细胞免疫荧光化学染色方法检测β-catenin和Gsk-3β的阳性细胞率.利用红景天苷分别诱导MSCs 1,2,8,12,24,48和72 h后,利用实时PCR技术检测Wnt/β-catenin信号通路的关键信号分子wnt3a、Axin2、Lrp6和Gsk-3β mRNA的表达;采用Western blot方法分析D1细胞诱导12、24、48和72 h后,β-catenin和Gsk-3β蛋白的表达;运用Wnt/β-catenin信号通路特异性阻断剂DKK1阻断Wnt/β-catenin信号通路,Western blot方法分析红景天苷对β-catenin和NSE蛋白表达的影响.结果:红景天苷诱导24 h时β-catenin 的阳性率可达55.76 %,与其他组和对照组比较差异具有统计学意义(P<0.01),诱导24 h后Gsk-3β的阳性率与其他时间和对照组比较差异有统计学意义(P<0.05).实时PCR检测结果显示,红景天苷诱导MSCs不同时间能促进Wnt/β-catenin信号通路中关键信号分子Wnt3a、Axin2、Lrp6和Gsk-3β mRNA的表达,诱导不同时间Wnt3a、Axin2、Lrp6和Gsk-3β mRNA的表达不尽相同.Western blot结果表明,红景天苷诱导D1细胞12 h和24 h时β-catenin蛋白的表达明显上调,且与其他组比较差异具有统计学意义(P<0.05);随着作用时间的延长,Gsk-3β蛋白的表达增加且差异具有统计学意义(P<0.05),阻断Wnt/β-catenin信号通路后,β-catenin和NSE蛋白的表达水平明显下调.结论:红景天苷能诱导D1细胞定向分化为神经元样细胞,红景天苷通过激活Wnt/β-catenin信号通路实现其诱导MSCs向神经细胞定向分化.%Objective: This work was carried out to investigate the directed differentiation of MSCs into neural cells in Wnt/β-catenin signaling pathway induced by salidroside ( SD ), utilizing the mouse bone marrow-derived mesenchymal stem cell series D1 as the model. Methods:Two groups were designed,a control group ( D/F12 complete medium ) and a SD group ( 100 μg/mL + D/F12 complete medium ). These two groups were left to induction for 12,24,48 and 72 h and the positive rate of NSE,MAP2, (3-Tubulin Ⅲ and GFAP,as well as the expression of β-catenin and GSK-3 β were detected by immunocytochemistry staining method. The real-time PCR results gave the expression of the critical signaling molecules in Wnt/β-catenin signaling pathway,such as wnt3a, Axin2,Lrp6 and Gsk-3 β mRNA. The expression of β-catenin and Gsk-3 β were analyzed according to Western blot method. Results: It was found that salidrosides can induce MSCs differentiate into neuron-like cells. In the case of SD group with the induction time of 24 h,the positive rate of β-catenin was 55. 76% and a significant difference was observed compared with the control group ( P < 0. 01 ). Meanwhile, the positive rate of Gsk-3 β also exhibited significant difference when induced for a period of 24 and 48 h. Realtime PCR results indicated that all the signaling molecules, Wnt3a, Axin2, Lrp6, and Gsk-3 β mRNA could be efficiently expressed in the SD groups at each time point. The only difference is that the extent of expression changed with the increasing induction time. From the Western blot,the expression of β-catenin protein and Gsk-3β gradually went up in the SD groups with the increasing time, which is significantly different from the control groups ( P <0. 05 ). The expression of β-catenin and NSE protein, which was induced with salidroside, were significantly decreased while Wnt/β-catenin signal pathway was blocked by DKK1. Conclusions: Salidroside can induce the differentiation of MSCs into neuron-like cells and Wnt/β-catenin signaling pathway could be activated by SD.