摘要
目的:探讨不同浓度的淫羊藿苷对体外培养的骨质疏松大鼠骨髓间充质干细胞(MSCs)成脂与成骨双向分化的作用及其机制。方法:运用全骨髓贴壁法分离纯化和培养Sprague Dawley大鼠MSCs,免疫荧光检测MSCsy阳性标志CD44和CD90;CCK-8检测不同浓度淫羊藿苷对MSCs细胞增殖作用影响;观察不同浓度淫羊藿苷对MSCs成脂和成骨分化的影响;qRT-PCR和Western blot检测给药前后MSCs内成脂特异性基因PPARγ、Adipsin和FABP4以及成骨特异性基因RUNX2、ALP和OPN的变化。Western blot检测淫羊藿苷刺激对Wnt/β-catenin通路激活的影响。结果:免疫荧光结果显示MSCs阳性标志CD44和CD90均表现出红色荧光遍布胞质;CCK-8检测结果显示10、20、30和40 ng/ml淫羊藿苷在1~4 d对MSCs增殖均无影响,50 ng/ml淫羊藿苷在第3~4 d时明显抑制MSCs增殖(P<0.05);与空白对照组相比,10 ng/ml淫羊藿苷能抑制MSCs向成脂方向分化,同时显著下调成脂特异性基因PPARγ、Adipsin和FABP4的表达(P<0.05),而40 ng/ml淫羊藿苷作用则与之相反;10 ng/ml淫羊藿苷能促进MSCs向成骨方向分化,同时显著上调成骨特异性基因RUNX2、ALP和OPN的表达以及OC的活性,而40 ng/ml淫羊藿苷作用则与之相反。10 ng/ml和40 ng/ml淫羊藿苷均显著上调Wnt/β-catenin信号通路β-catenin和Wnt7(P<0.05)表达,同时下调GSK-3β的表达(P<0.05)。结论:不同浓度的淫羊藿苷通过激活Wnt/β-catenin信号通路促进RUNX2、ALP和OPN的表达或者抑制PPARγ、Adipsin和FABP4的表达从而促进大鼠MSCs成骨分化,抑制其成脂分化作用。
Objective:To investigate the effects of different concentrations of icariin on the adipogenic and osteogenic differentiation of osteoporosis rat bone marrow mesenchymal stem cells(MSCs) in vitro and its mechanism.Methods:Purification and culture of Sprague Dawley rat MSCs by whole bone marrow adherence method,immunofluorescence detection of MSCsy positive markers CD44 and CD90;CCK-8 detected the effects of different concentrations of icariin on MSCs cell proliferation;observed the effects of icariin at different concentrations on adipogenic and osteogenic differentiation of MSCs;qRT-PCR and Western blot were used to detect the changes of adipogenic specific genes PPARγ,Adipsin and FABP4 and osteogenic specific genes RUNX2,ALP and OPN in MSCs before and after administration.And Western blot detect the effect of icariin stimulation on Wnt/β-catenin pathway activation.Results:The results of CCK-8 showed that 10 ng/ml,20 ng/ml,30 ng/ml and 40 ng/ml icariin had no effect on the proliferation of MSCs in 1-4 days,50 ng/ml icariin at On day 3-4,the proliferation of MSCs was significantly inhibited(P<0.05).Compared with the blank control group,10 ng/ml icariin inhibited the differentiation of MSCs into adipogenic phase,and significantly down-regulated the lipid-specific genes PPARγ,Adipsin and FABP4(P<0.05),while the effect of 40 ng/ml icariin was opposite.10 ng/ml icariin could promote the differentiation of MSCs into osteogenesis,and significantly up-regulate the expression of osteogenesis-specific genes RUNX2,ALP and OPN and the activity of OC,while the effect of 40 ng/ml icariin in contrast.The icariin(10 ng/ml and 40 ng/ml)significantly up-regulated the expression of Wnt/β-catenin signaling pathway β-catenin and Wnt7(P<0.05),and down-regulated the expression of GSK-3β(P<0.05).Conclusion:Icariin promotes the expression of RUNX2,ALP and OPN by activating Wnt/β-catenin signaling pathway,inhibits the expression of PPARγ,Adipsin and FABP4,and promotes osteogenic differentiation of rat MSCs and inhibits its adipogenic differentiation.
作者
李智奎
孔俊博
赵王林
LI Zhi-Kui;KONG Jun-Bo;ZHAO Wang-Lin(Department of Orthopaedics,Yunnan Provincial Hospital of Traditional Chinese Medicine,Kunming 650021,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第24期2985-2990,共6页
Chinese Journal of Immunology
基金
云南省应用基础研究重点项目(2013FZ194)
关键词
淫羊藿苷
骨质疏松
MSCS
成脂分化
成骨分化
Icariin
Osteoporosis
MSCs
Adipogenic differentiation
Osteogenic differentiation
