感觉神经肽P物质激活Wnt/β-catenin信号通路促进间充质干细胞增殖的实验研究  

Neuropeptide SP activates the Wnt/β-catenin signaling pathway and enhances the proliferation of bone marrow mesenchymal stem cells

作　　者：喻伟[1] 程鹏[1] 黄晖[1] 黄鑫[1] 陈安民[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《骨科》2015年第2期62-65,共4页ORTHOPAEDICS

基　　金：国家自然科学基金资助项目(No.81171696);湖北省医学领军人才培养工程专项经费资助项目

摘　　要：目的探讨感觉神经肽P物质(substance P,SP)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响并初步揭示其中可能的机制。方法体外培养SD(Sprague-Dawley)大鼠BMSCs。细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)法检测不同浓度SP刺激下的BMSCs增殖活性;在10-8 mol/L的SP刺激下,采用实时荧光定量PCR(real-time qPCR,RT-qPCR)检测第1、3、5和7天时Wnt/β-catenin信号通路相关基因(β-catenin、c-myc、cyclin D)的表达情况。SP组单纯添加10-8 mol/L的SP,SP+LiCl(糖原合成激酶-3的抑制剂LiCl)组在SP组基础上添加6×10-3 mol/L的LiCl,SP+Dkk-1(Dickkopf-1)组在SP组基础上添加10-9 mol/L的Dkk-1,以添加等量聚丁烯琥珀酸酯(poly butylenes succinate,PBS)为空白对照组。结果 CCK-8法显示SP明显促进BMSCs增殖,以10-8 mol/L的SP作用最为显著。RT-qPCR结果显示SP刺激下,β-catenin、c-myc、cyclin D的mRNA表达有显著差异,10-8 mol/L的浓度差异最为显著,并且呈明显的时间依赖,第5天mRNA表达出现顶峰。并且SP+LiCl组β-catenin、c-myc、cyclin D的mRNA和蛋白水平显著增强,而SP+Dkk-1组明显抑制。结论 SP通过激活Wnt/β-catenin通路而显著促进BMSCs增殖。Objective To explore the effects of neuropeptide substance P（SP）on proliferation of bone marrow mesenchymal stem cells（BMSCs）and reveal the potential mechanism.Methods BMSCs of SD rats were cultured at 37 ℃ and 5% CO2.The proliferative ability of BMSCs with different doses of SP was measured by CCK-8 kit.The expression of the related genes of the Wnt/β-catenin signaling pathway,such asβ-catenin,c-myc,cyclin D,was detected by RT-qPCR in the dose-course and time-course.In addition,the agonist LiCl and the inhibitor Dkk-1 of the Wnt/β-catenin signaling pathway were separately used and the expression ofβ-catenin,c-myc,and cyclin D mRNA and proteins was detected as well.Results The BMSCs staining was typical with alizarin red staining after inducion of osteogenesis,oil red O staining after induction of adipogenesis,and alcian blue staining after induction of chondrogenesis.The BMSCs treated different doses of SP,especially 10-8 mol/L,had a greater proliferative ability than the controls.The mRNA expression ofβ-catenin,c-myc,and cyclin D was significantly different in SP dose-course and time-course,especially in 10-8 mol/L after 5 days.The expression levels of related genes to the Wnt/β-catenin signaling pathway were up-regulated by LiCl,and down-regulated by Dkk-1.Conclusion Neuropeptide SP can enhance the proliferation of BMSCs probably by regulating the Wnt/β-catenin signaling pathway.

关 键 词：感觉神经肽P物质 骨髓间充质干细胞 增殖 WNT/Β-CATENIN信号通路 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]