Proteomic Analysis Of Rat Chondrocytes Under Periodic Mechanical Stress

Part ? In search of the differential expressed proteins of rat chondrocytes under periodic mechanical stressObjectiveTo screen the differential expressed proteins of rat chondrocytes under periodic mechanical stress using quantitative proteomic analysis.MethodsRat chondrocytes were isolated and cultured in vitro and then divided into control group and pressure group.The control group were cultured under static condition,the pressure group were subjected to stress varying from 0 to 200 KPa at 0.1 Hz for 8 hours per day,lasting 3 days.Quantitative real-time PCR analysis was used to observed the expression of aggrecan and collagen?.Cell counting and the CCK-8 assay were used to detect the proliferation of chondrocytes.The total proteins of two groups were extracted and labeled by isobaric tags for relative and absolute quantitation(iTRAQ)respectively.Then the labeled proteins were isolated by liquid chromatography and identified by Q-Exactive mass spectrometry.After the processes mentioned above,we used bioinformatics analysis to compare the differentially expressed proteins.ResultsA total of 5468 proteins were identified in our study.The differentially expressed proteins were 485,among which 168 were up-regulated and 317 were down-regulated.The bioinformatics analysis indicated that 478 proteins were involved in 6079 GO(Gene Ontology)function entries and 230 proteins participated in 241 KEGG(Kyoto Encyclopedia of Genes and Genomes)signal pathways.We also select Grb2 as the target protein of our further research according to the bioinformatics analysis.ConclusionWe have successfully constructed the differentially expressed proteome profiles of rat chondrocytes under periodic mechanical stress and static conditions.This profiles will inspire the further study on the mechanism underlying the ability of chondrocytes to sense and respond to periodic mechanical stress.Part ? The roles of Grb2 in periodic mechanical stress-induced rat chondrocyte mitogenic effects ObjectiveTo explore the roles of Grb2 in periodic mechanical stress-induced rat chondrocyte mitogenic effects.MethodsWe used the lentivirus vectors of Lv-Grb2 or Lv-Grb2-RNAi to transfact the chondrocytes in order to up-regulate or down-regulate the expression of Grb2.Three days cultured under the condition of 37? and 5%CO2 after pretreatment with lentivirus vectors,green fluorescence of rat chondrocytes were detected by fluorescence microscope and the expression of Grb2 were detected by real-time PCR and Western blot.Two groups of Lv-Grb2 and Lv-Grb2-RNAi were cultured under static conditions or periodic mechanical stress for 8 h per day,lasting 3 days,prior to direct cell counting and the CCK-8 assay,respectively.Quantitative real-time PCR analysis,immunofluorescence and immunohistochemistry were used to observed the expression of aggrecan and collagenII.Western blot was used to detected the phosphorylation of FAK-Tyr397?FAK-Tyr576/577 and ERK1/2.Results1.We evaluated GFP expression in each group by fluorescence microscope.More than 80%of chondrocytes were effectively transfected suggesting a high transfection efficiency.Western blot results suggest Lv-Grb2 enhances the expression of Grb2 and Lv-Grb2-RNAi suppress its expression.2.Up-regulation of Grb2 promote periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis,while down-regulation has the opposite effect.Up-regulating Grb2 under the condition of periodic mechanical stress can facilitate the phosphorylation of FAK-Tyr397?FAK-Tyr576/577 and ERK1/2.ConclusionThis research demonstrate the key role of Grb2 in the cellular responses of periodic mechanical stress on chondrocytes.It can promotes Integrin-induced FAK-Tyr397 and FAK-Tyr576/577 phosphorylation and directs chondrocytes proliferation and matrix synthesis under the condition of stress.