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浙江大学学报(医学版)  2011, Vol. 40 Issue (6): 630-640    DOI: 10.3785/j.issn.1008-9292.2011.06.010
论著     
Wnt/β-catenin信号通路对间充质干细胞衰老的影响及其作用机制
项晓霞,陈律,王骏浩,张余斌,张大勇
浙江大学城市学院医学与生命科学学院,浙江 杭州 310015
Role of Wnt/β-catenin signaling in aging of mesenchymal stem cells of rats
XIANG Xiao-xia,CHEN Lü,WANG Jun-hao,ZHANG Yu-bin,ZHANG Da-yong
School of Medicine and Life Science,Zhejiang University City College,Hangzhou 310015,China
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摘要: 目的:研究不同年龄血清对Wnt/β-catenin信号通路激活水平的影响,探讨Wnt/β-catenin信号通路对间充质干细胞(MSCs)衰老、增殖、存活能力的作用及其机制。
方法:提取年轻(8~12 周)及老年(64~72 周)SD大鼠血清,实验分为4组,分别为年轻血清(Young rat serum,YRS)组,YRS+Wnt 3a组、老年血清(Old rat serum,ORS)组和ORS+DKK1组。免疫细胞化学染色和Western blotting检测胞浆、胞核β-catenin的表达。SA-β-半乳糖苷酶染色检测各组细胞衰老,MTT法检测细胞增殖,AO/EB染色法检测MSCs存活和凋亡。为研究Wnt/β-catenin信号通路导致MSCs衰老的作用机制,免疫细胞化学染色和Western blotting检测γ-H2A.X和p53蛋白表达,RT-PCR法检测p53、p21 mRNA表达。
结果:ORS组β-catenin表达较YRS组明显增强,尤其是胞核,出现β-catenin聚集现象,而ORS+DKK1组β-catenin表达减弱。与YRS组相比,YRS+Wnt 3a组细胞SA-β-半乳糖苷酶染色阳性细胞数显著增多(P<0.01),细胞增殖和存活能力减弱。而ORS+DKK1组与ORS组相比,SA-β-半乳糖苷酶染色阳性细胞数显著减少(P<0.01),细胞增殖和存活能力增强。免疫细胞化学染色、Western blotting和RT-PCR检测结果显示,ORS组内γ-H2A.X、p53、p21表达较YRS组明显增强,而在ORS内加入DKK1后,γ-H2A.X、p53、p21表达较ORS组明显减弱。
结论:ORS培养可以激活MSCs内Wnt/β-catenin信号通路。Wnt/β-catenin信号通路对MSCs衰老发挥重要调控作用。DNA损伤反应和p53/p21途径是Wnt/β-catenin信号通路导致MSCs衰老的重要介导因素。
关键词: 间质干细胞/病理学 细胞增殖 信号传导 Wnt/&beta-catenin信号通路 骨髓间充质干细胞 血清 衰老 DNA损伤反应 p53/p21途径    
Abstract: Objective: To investigate the role of Wnt/β-catenin signaling in aging of mesenchymal stem cells (MSCs) of rats.
Methods: Serum samples were collected from young (8~12 w) and aged (64~72 w) SD rat.Four experiment groups were assigned:young rat serum (YRS),YRS+Wnt 3a,old rat serum (ORS) and ORS + DKK1 groups.Immunofluorescence and Western blotting were used to detect the expression of intracellular β-catenin.The senescence-associated changes were examined with SA-β-galactosidase staining.The proliferation ability was tested by MTT assays.The survived and apoptotic cells were determined by AO/EB staining.The expressions of γ-H2A.X and p53 protein were detected by immunofluorescence and Western blotting.RT-PCR was used to detect the expression of p53 and p21 mRNA.
Results: Compared with the YRS group,the intracellular expression of β-catenin in the ORS group was significantly increased,especially in the nuclei of MSCs.After treatment of DKK1 in ORS,the β-catenin expression was reduced.The number of SA-β-galactosidase positive MSCs was significantly higher in the YRS+Wnt 3a group than that in the YRS group (P<0.01),and the proliferative and survival ability of MSCs was significantly decreased in the YRS+Wnt 3a group.The number of SA-β-galactosidase positive MSCs in the ORS+DKK1 group was significantly decreased compared with that in ORS group (P<0.01),and the proliferative and survival ability of MSCs was significantly increased in the ORS+DKK1 group.The expression of γ-H2A.X,p53 and p21 was markedly increased in the ORS group than that in YRS group,however,after treatment with Wnt/β-catenin signaling inhibitor DKK1,the expression of γ-H2A.X,p53 and p21 was significantly decreased compared with that in the ORS group.
Conclusions: Results suggest that the Wnt/β-catenin signaling is activated in the MSCs cultured with ORS and excessive activation of Wnt/β-catenin signaling can promote MSCs aging.The DNA damage response and p53/p21 pathway may be main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling.
Key words: Mesenchymal stem cells/pathol    Cell proliferation    Signal transduction    Wnt/β-catenin signaling    Mesenchymal stem cells (MSCs)    Aging    serum    DNA damage response    p53/p21 pathway
出版日期: 2011-11-25
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项晓霞;陈律;王骏浩;张余斌;张大勇. Wnt/β-catenin信号通路对间充质干细胞衰老的影响及其作用机制[J]. 浙江大学学报(医学版), 2011, 40(6): 630-640.

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https://www.zjujournals.com/med/CN/Y2011/V40/I6/630

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