牛膝醇提物诱导兔骨髓间充质干细胞软骨分化的蛋白组学分析

Proteomic analysis on cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract

背景:前期研究发现牛膝醇提物具有诱导骨髓间充质干细胞定向软骨细胞分化的作用,但具体作用蛋白靶点和网络机制不详。目的:观察牛膝醇提物诱导兔骨髓间充质干细胞软骨分化的蛋白质组学分析及蛋白质相互作用网络构建。方法:采用密度梯度离心联合细胞贴壁法分离培养新西兰大白兔骨髓间充质干细胞,取第3代细胞随机分成5组:空白组、对照组、牛膝醇提物低、中、高剂量组,连续成软骨诱导培养21 d后,用qRT-PCR检测Ⅱ型胶原mRNA表达及甲苯胺蓝染色鉴定软骨细胞形成,应用绝对定量同位素标记(iTRAQ)结合双向液相色谱-串联质谱(2DLC-MS/MS)技术对各组差异表达蛋白质进行鉴定,并对差异蛋白进行GO分析、KEGG分析及蛋白质网络相互作用分析。结果与结论:(1)qRT-PCR结果显示牛膝醇提物高剂量组较其他组Ⅱ型胶原mRNA表达明显增高(P<0.05),牛膝醇提物高剂量组甲苯胺蓝染色阳性,蛋白组学分析共鉴定到1354个差异蛋白点,上调蛋白633个,下调蛋白721个。(2)根据qRT-PCR结果对空白组、牛膝醇提物高剂量组、对照组3组差异表达蛋白进行生物信息分析。GO分析发现这些差异表达蛋白参与代谢、细胞分化、细胞周期与凋亡、炎症反应、免疫调控、氧化应激、磷酸化、泛素化、癌相关等;KEGG分析获得与骨关节病最相关的10个典型信号通路:白细胞介素17信号通路,Toll样受体信号通路,Wnt信号通路,PI3K-Akt信号通路,mTOR信号通路,Jak-STAT信号通路,NF-kappa B信号通路,MAPK信号通路,AMPK信号通路,HIF-1信号通路;根据组间差异蛋白,使用Cytoscape3.6.0软件成功构建蛋白互作网络图。(3)结果表明,牛膝醇提物可以通过调控氧化、细胞周期与凋亡、细胞结构改变、细胞分化、代谢及炎症损伤等环节诱导兔骨髓间充质干细胞成软骨分化,具有多靶点、多中心的网络调控作用,其具体作用机制有待进一步研究。

BACKG ROUND:Preliminary studies have found that Achyranthes bidentata alcohol extra ct can induce the directional chondrocyte differentiation of bone marrow mesenchymal stem cells,but the specific protein targets and network mechanisms are unknown.OBJECTIVE:To observe the proteomic analysis of ca rtilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extra ct and the construction of protein interaction network.METHODS:Bone marrow mesenchymal stem cells of New Zealand white rabbits were isolated and cultured by density gradient centrifugation combined with bone marrow adherent method.Passage 3 cells were randomly divided into five groups:blank group,control group,low-dose Achyranthes bidentata alcohol extract group,medium-dose Achyranthes bidentata alcohol extract group,and high-dose Achyranthes bidentata alcohol extract group.After continuous induction and culture for 21 days,qRT-PCR was used to detect the expression of typeⅡcollagen mRNA and toluidine blue staining was used to identify the formation of chondrocytes.The differentially expressed proteins of different groups were identified by absolute quantitative isotope labeling(iTRAQ)combined with two-way liquid chromatography to ndem mass spectrometry,and the differentially expressed proteins were analyzed by GO analysis,KEGG analysis and protein interaction network analysis.RESULTS AND CONCLUSION:(1)The qRT-PCR results showed that the expression of typeⅡcollagen mRNA in the high-dose Achyranthes bidentata alcohol extract group was significantly higher than that in other groups(P<0.05),and toluidine blue staining displayed positive reaction in the high-dose Achyranthes bidentata alcohol extract group.A total of 1354 differential protein spots were identified by proteomic analysis,including 633 up-regulated proteins and721 down-regulated proteins.(2)According to the res ults of qRT-PCR,the differentially expressed proteins of blank group,high-dose Achyranthes bidentata alcohol extract group and control group were analyzed.Go analysis showed that these differentially expressed proteins were involved in metabolism,cell differentiation,cell cycle and apoptosis,inflammato ry response,immune regulation,oxidative stress,phosphorylation,ubiquitination,cancer and so on.KEGG analysis obtained 10 typical signal pathways most related to osteoarthritis:interleukin-17 signaling pathway,Toll like receptor signaling pathway,Wnt signaling pathway,PI3K-Akt signaling pathway,mTOR signaling pathway,JAK-STAT signaling pathway,NF-kappa B signaling pathway,MAPK signaling pathway,AMPK signaling pathway,and HIF-1 signaling pathway.According to the protein difference between groups,Cytoscape 3.6.0 software was used to successfully construct protein interaction network diagram.(3)These res ults suggest that Achyranthes bidentata alcohol extract can induce chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells by regulating oxidation,cell cycle and apoptosis,changes in cell structure,cell differentiation,metabolism and inflammatory injury.It has multi-target and multi-center network regulation,and its specific mechanism needs to be further studied.