| 摘要: |
| 目的 探讨初级纤毛介导的Sonic hedgehog(Shh)信号对大鼠骨髓基质细胞(MSCs)神经元样细胞分化的影响。方法 全骨髓贴壁法分离培养大鼠MSCs。实验分为白藜芦醇正常培养组、白藜芦醇诱导组、Smoothened (Smo)激动剂SAG组、Smo抑制剂环巴明组,分别给予相应的药物处理。倒置显微镜下观察各组细胞形态; 免疫荧光法检测各组细胞初级纤毛及Shh信号通路相关蛋白\[Ac-Tu、Patched (Ptc)、Smo、Gli1\]的表达; RT-PCR检测各组细胞Smo、Gli1 mRNA的表达;蛋白质印迹法检测各组细胞Smo和Gli1蛋白的表达。结果 正常培养的大鼠MSCs不表达初级纤毛。经过预诱导或饥饿处理24 h后,MSCs表达初级纤毛及Ptc、Smo和Gli1蛋白,其中Smo和Gli1蛋白位于胞质,Ptc蛋白位于初级纤毛。正常培养时,白藜芦醇不能促使Smo移位及MSCs向神经元样细胞分化。当MSCs表达初级纤毛时,白藜芦醇及SAG可使Smo从胞质进入初级纤毛,同时伴随MSCs向神经元样细胞分化及 Smo、Gli1 mRNA和蛋白表达水平的增加(P<0.05); 加入抑制剂环巴明后,Smo仍主要表达于胞质,Smo、Gli1 mRNA和蛋白的表达水平降低(P<0.05),MSCs向神经元样细胞的分化受抑制。结论 MSCs表达初级纤毛并存在Shh信号,初级纤毛介导的Shh信号参与调控大鼠MSCs的神经元样细胞分化。 |
| 关键词: Shh信号 初级纤毛 骨髓基质细胞 神经元样细胞 细胞分化 |
| DOI： |
| 投稿时间：2013-03-18修订日期：2013-08-20 |
| 基金项目:国家自然科学基金面上项目(81071119),重庆市卫生局科研基金(2009-2-359). |
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| Effect of primary cilia-mediated Shh signaling on neuronal-like cell differentiation of rat bone marrow stromal cells |
| HUANG Jia-gui,XU Lan,SHEN Chang-bo,LIU Shu,YANG Qin* |
(Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
*Corresponding author.) |
| Abstract: |
| Objective To study the effect of primary cilia-mediated sonic hedgehog (Shh) signaling on differentiation of rat bone marrow stromal cells (MSCs) into neuron-like cells. Methods Rat MSCs were isolated and cultured. The study was divided into normal resveratrol-cultured group, resveratrol-induced group, SAG (Smoothened \[Smo\]agonist) group, and cyclopamine (Smo inhibitor) group. Cell morphology was observed under inverted microscope; the expressions of Ac-Tu, Ptc, Smo, and Gli1 were examined by immunofluorescence method. Western blotting analysis was used to detect the protein expressions of Smo and Gli, and RT-PCR was applied to detect mRNA expressions of Smo and Gli1. Results No expression of primary cilia was found in the normally cultured MSCs. After pre-induction or 24 h starvation, MSCs expressed primary cilia, Ptc, Smo and Gli1 proteins, with Ptc protein in the primary cilia and Smo, Gli1 in the cytoplasm. In normal cultivation, resveratrol did not promote the translocation of Smo or induce differentiation of MSCs into neuronal-like cells. When MSCs expressed primary cilia, resveratrol and SAG led to translocation of Smo from the cytoplasm into primary cilia, accompanied by MSCs differentiation into neuronal-like cells and significantly increased expression of Smo and Gli1 mRNA and protein (P＜0.05). When cyclopamine was added, Smo remained expressed in the cytoplasm, the expression of Smo and Gli1 mRNA and protein was significantly decreased (P＜0.05), and MSCs differentiation into neuronal-like cells was inhibited. Conclusion MSCs express primary cilia and have Shh signaling. Primary cilia-mediated Shh signaling participates in MSCs differentiation into neuronal-like cells. |
| Key words: Shh signaling primary cilia bone marrow stromal cells neuronal-like cells cell differentiation |
