周期性机械应力条件下大鼠软骨细胞的蛋白质组学分析
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摘要
目的应用定量蛋白质组学技术筛选周期性机械应力条件下大鼠软骨细胞差异表达蛋白质。方法将体外培养的大鼠软骨细胞随机分为对照组和周期性机械应力组,分别提取细胞蛋白,并依次进行同位素标记、液相色谱分离、质谱鉴定和生物信息学分析。结果共鉴定出5468个蛋白,差异性表达蛋白为485个;其中上调168个,下调317个。经生物信息学分析,共有478个蛋白被6079条基因本体论(GO)功能条目注释,230个蛋白参与了241条京都基因与基因组百科全书(KEGG)信号通路。结论成功构建周期性机械应力条件下大鼠软骨细胞差异蛋白质表达谱,为周期性机械应力促进大鼠软骨细胞增殖和细胞外基质合成的机制研究提供实验基础。
Objective To screen the differentially expressed proteins of rat chondrocytes under periodic mechanical stress by quantitative proteomics technique.Methods Rat chondrocytes were cultured in vitro and then divided into two groups of A(normal control)and B(under periodic mechanical stress).The total proteins in groups of A and B were extracted and labeled by isobaric tags for relative and absolute quantitation,respectively.Then the labeled proteins were isolated by liquid chromatography,identified by Q-exactive mass spectrometry and analyzed by bioinformatics for comparing the differentially expressed proteins.Results A total of 5468 proteins was identified.The differentially expressed proteins were 485,among which 168 proteins were upregulated and 317 proteins were downregulated.The bioinformatics analysis indicated that 478 proteins were involved in6079 gene ontology(GO)function entries and 230 proteins were participated in 241 kyoto encyclopedia of genes and genomes(KEGG)signal pathways.Conclusion The differentially expressed proteome profile of rat chondrocytes under periodic mechanical stress has been successfully constructed,which may provide a foundation for studying the underlying mechanism of proliferation of rat chondrocytes and synthesis of extracellular matrix promoted by periodic mechanical stress.
Objective To screen the differentially expressed proteins of rat chondrocytes under periodic mechanical stress by quantitative proteomics technique.Methods Rat chondrocytes were cultured in vitro and then divided into two groups of A(normal control)and B(under periodic mechanical stress).The total proteins in groups of A and B were extracted and labeled by isobaric tags for relative and absolute quantitation,respectively.Then the labeled proteins were isolated by liquid chromatography,identified by Q-exactive mass spectrometry and analyzed by bioinformatics for comparing the differentially expressed proteins.Results A total of 5468 proteins was identified.The differentially expressed proteins were 485,among which 168 proteins were upregulated and 317 proteins were downregulated.The bioinformatics analysis indicated that 478 proteins were involved in6079 gene ontology(GO)function entries and 230 proteins were participated in 241 kyoto encyclopedia of genes and genomes(KEGG)signal pathways.Conclusion The differentially expressed proteome profile of rat chondrocytes under periodic mechanical stress has been successfully constructed,which may provide a foundation for studying the underlying mechanism of proliferation of rat chondrocytes and synthesis of extracellular matrix promoted by periodic mechanical stress.
引文
[1]Wolf A,Ackermann B,Steinmeyer J.Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading[J].Cell Tissue Res,2007,327(1):155-166.
[2]Akimoto T,Kawanishi M,Ushida T.Mechanical stress and tissue engineering[J].Clin Calcium,2008,18(9):1313-1320.
[3]Kock LM,Schulz RM,van Donkelaar CC,et al.RGDdependent integrins are mechanotransducers in dynamically compressed tissue-engineered cartilage constructs[J].J Biomech,2009,42(13):2177-2182.
[4]Liang W,Ren K,Liu F,et al.Periodic mechanical stress stimulates the FAK mitogenic signal in rat chondrocytes through ERK1/2activity[J].Cell Physiol Biochem,2013,32(4):915-930.
[5]Sandberg A,Lindell G,Kllstrm BN,et al.Tumor proteomics by multivariate analysis on individual pathway data for characterization of vulvar cancer phenotypes[J].Mol Cell Proteomics,2012,11(7):M112.016998.
[6]Bougault C,Aubert-Foucher E,Paumier A,et al.Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes[J].PLoS One,2012,7(5):e36964.
[7]Price PA,Caputo JM,Williamson MK.Bone origin of the serum complex of calcium,phosphate,fetuin,and matrix Gla protein:biochemical evidence for the cancellous boneremodeling compartment[J].J Bone Miner Res,2002,17(7):1171-1179.
[8]Vyner MC,Amsden BG.Polymer chain flexibility-induced differences in fetuin A adsorption and its implications on cell attachment and proliferation[J].Acta Biomater,2016,31:89-98.
[9]Ren K,Liu F,Huang Y,et al.Periodic mechanical stress activates integrinβ1-dependent Src-dependent PLCγ1-independent Rac1mitogenic signal in rat chondrocytes through ERK1/2[J].Cell Physiol Biochem,2012,30(4):827-842.
[10]张旻,王美青,王景杰.G蛋白及蛋白激酶C在髁突软骨细胞力学信号转导过程中的作用[J].实用口腔医学杂志,2005,21(2):163-166.
[11]Lee KJ,Yoo YH,Kim MS,et al.CD99inhibits CD98-mediatedβ1integrin signaling through SHP2-mediated FAK dephosphorylation[J].Exp Cell Res,2015,336(2):211-222.
[2]Akimoto T,Kawanishi M,Ushida T.Mechanical stress and tissue engineering[J].Clin Calcium,2008,18(9):1313-1320.
[3]Kock LM,Schulz RM,van Donkelaar CC,et al.RGDdependent integrins are mechanotransducers in dynamically compressed tissue-engineered cartilage constructs[J].J Biomech,2009,42(13):2177-2182.
[4]Liang W,Ren K,Liu F,et al.Periodic mechanical stress stimulates the FAK mitogenic signal in rat chondrocytes through ERK1/2activity[J].Cell Physiol Biochem,2013,32(4):915-930.
[5]Sandberg A,Lindell G,Kllstrm BN,et al.Tumor proteomics by multivariate analysis on individual pathway data for characterization of vulvar cancer phenotypes[J].Mol Cell Proteomics,2012,11(7):M112.016998.
[6]Bougault C,Aubert-Foucher E,Paumier A,et al.Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes[J].PLoS One,2012,7(5):e36964.
[7]Price PA,Caputo JM,Williamson MK.Bone origin of the serum complex of calcium,phosphate,fetuin,and matrix Gla protein:biochemical evidence for the cancellous boneremodeling compartment[J].J Bone Miner Res,2002,17(7):1171-1179.
[8]Vyner MC,Amsden BG.Polymer chain flexibility-induced differences in fetuin A adsorption and its implications on cell attachment and proliferation[J].Acta Biomater,2016,31:89-98.
[9]Ren K,Liu F,Huang Y,et al.Periodic mechanical stress activates integrinβ1-dependent Src-dependent PLCγ1-independent Rac1mitogenic signal in rat chondrocytes through ERK1/2[J].Cell Physiol Biochem,2012,30(4):827-842.
[10]张旻,王美青,王景杰.G蛋白及蛋白激酶C在髁突软骨细胞力学信号转导过程中的作用[J].实用口腔医学杂志,2005,21(2):163-166.
[11]Lee KJ,Yoo YH,Kim MS,et al.CD99inhibits CD98-mediatedβ1integrin signaling through SHP2-mediated FAK dephosphorylation[J].Exp Cell Res,2015,336(2):211-222.