Proteomincs And UPS Mechanisms Of Mouse Embryonic Stem Cells Directional Differentiation Into Cardiomyocytes Induced By Icariin In Vitro

Embryonic stem cells can be directed to differentiate into the typical structure and function of myocardial cells.The differentiation system has become a important tool of studying cardiac gene expression and function.Currently,ES cells,directed to differentiate into myocardial cells during a number of gene function and signal transduction network control mechanism is not yet very clear.Although some studies conducted a preliminary exploration,but mainly concentrated in one or a few known genes and proteins of myocardial cells in terminal differentiation pathway.The directed differentiation of ES cells,there are many genes,transcription factors,proteins and growth factors at different levels and at different times regulates cardiac cell differentiation in the directed differentiation of ES cells.If only use the traditional gene and protein analysis methods,it is difficult to explain the development of the whole heart basic mechanisms systematic and thoroughly.Only from the DNA sequence is not yet the time to answer the expression of a gene expression level, protein post-translational processing and modification of the conditions,etc.So studying the overall level of protein expression and function has become increasingly important. Developed in recent years,proteomics technology is genome coding for the study of all proteins from the cellular level and the overall level studies in composition and changes of the protein,in-depth understanding of a variety of organisms,physiological and pathological processes.Its core technology,two-dimensional electrophoresis,mass spectrometry and proteomics informatics for the rapid development can complete extremely complex changes in protein function and cytokine research.It can be explored in depth at every stage of cardiac differentiation of the characteristic products of gene transcription and function of protein expression patterns if applied to the process of differentiation of ES cells in vitro studies to clarify and differentiation of embryonic heart early onset of the essence of life phenomena,which explore the stem cell differentiation mechanisms and applications of great significance.Ubiquitin-proteasome system(UPS) participate in vivo in many life activities, such as cell cycle and division,differentiation and development,neural networks, morphogenesis,cell surface receptors or ion channels in the regulation of protein amount of control.There literature suggests that ubiquitin can promote the differentiation of embryonic stem cells,while blocking the activity of the proteasome can only in the early stages of differentiation of ESCs only the transcription activation regions of the genome.There is also literature suggests that blocking the activity of the proteasome would lead to the activity of NF-κB inhibited.So we can say,UPS in ESCs during differentiation is a double-edged sword.There is literature suggests that UCH-L1 in the differentiation of neural progenitor cells play an extremely important role.Therefore,we have the reason to believe that ESCs to differentiate into cardiac myocytes in the process,it also played a role can not be ignored.UPS is an important way to in vivo degradation of proteins and its mechanism are:1) by covalent binding of ubiquitin to mark the target protein;2) the marker protein for proteasome degradation. The primary role of UCH-L1 is to complete the task of ubiquitin - a combination of protein hydrolysis,which make ubiquitin repeat use,so that the concentration of ubiquitin in vivo maintained at a certain level can be considered ubiquitin and UCH-L1 expression was positively related.In our experiment,UCH-L1 in protein expression, indicating the hydrolysis of ubiquitin monomers more,and thus a large number of differentiation-promoting role of ubiquitin is also stronger.Available from this experiment,in the differentiation of ESCs to cardiomyocytes in the process,UPS's two members,ubiquitin plays a dominant role,while the role of the proteasome and NF-κB is mainly related to access.Meanwhile,we also believe that,UPS has played for the directed differentiation of certain weeds.Studies have shown that specific DNA binding proteins-transcription factors are redox-sensitive signaling pathways regulate gene expression targets.Nuclear factor-κB (nuclear factor-κB,NF-κB) is subject to the impact of cell redox state of the main transcription factor.Under normal circumstances,most cells NF-κB and its inhibitor (IκB) protein,a combination of family members staying in the cytoplasm,does not have transcriptional activity.When the cell by external factors,such as cytokines and other stimuli,IκB autophosphorylation occurred and rapidly degraded,NF-κB and IκB dissociation,Exposed nuclear localization signal and translocation into the nucleus and regulation of target gene transcription and expression.Studies have shown that,NF-κB with a variety of cardiac differentiation-related gene expression,myocardial accompanied during the development of NF-κB p65,p50 subunit,IκB subunits,its overexpression can start ANF,BNP and other gene re-expression of embryonic heart. Preliminary studies in our laboratory was found,ICA role of the EBs after,IκB is rapidly phosphorylated,and its own rapid degradation.Then observed that NF-κB p65 nuclear translocation,indicating a differentiation process in the ICA to promote cardiac activated NF-κB signaling.Our research group studies have confirmed that icariin can significantly improve the differentiation rate of ES cells in vitro to differentiate into cardiac cell and differentiation phase significantly ahead of schedule,and have a good relationship between dose-effect and time-effect.And we initially explored that the ICA can promote the development of myocardial dependent genes(such as BMP2,GATA4, NKx2.5,α-MHC,MLC-2v andβ-AR) and cardiac-specific sarcomeric proteins(such asα-actinin and troponin T) The expression increased and was on the order of the phase.This suggests that ICA-induced cardiomyocyte differentiation-dependent gene expression is closely related with its development.But only from the perspective of genetic research is not enough,we must explore the feasibility of gene transcription and translation process of the protein in order to reveal the true essence of the phenomenon of life and law.Now it has not been reported in directed against the ES cells to differentiate into cardiac cells,in particular,drug-induced differentiation of proteomics.If building a cardiac cell differentiation protein differential expression patterns ite can be explored in depth at every stage of cardiac differentiation of the characteristic products of gene transcription and function of protein expression patterns and to clarify the heart of early onset and differentiation of embryonic life of the nature of the phenomenon of great significance.Aim:To establish the small molecular weight protein expression profiles of mouse embryonic stem cells(ES cells) differentiating into cardiomyocytes induced by icariin (ICA) in vitro.And we selected the ubiquitin-proteasome system in identified proteins to explore the mechanisms of ICA-induced cadiomyocytes differentiation.Methods:Using the hanging drop culture method to differentiate ES cells in vitro into cardiomyocytes by ICA in vitro.Immunocytochemical was used to identify myocardial cells in order to determine whether there sarcomere comes from the ES cells to differentiate whether the myocardial cells.With 12.5%of two-dimensional gel electrophoresis separation of small molecules,proteins,stained using silver staining, scanned and image analysis to identify the different expression of differentiation-phase there is significant differences in protein spots,or the same split-phase induction group and spontaneous differentiation of ICA group,significant differences in expression of protein spots stained with Coomassie brilliant blue after the MS identification,and used RT-PCR,and immunofluorescence confirmed chemical method.In the identified proteins,select the ubiquitin-proteasome system as a mechanism for the study.Join proteasome inhibitor Epoxomicin,statistical differentiation rate of myocardial cells to detect the proteasome in the ES cells to differentiate into myocardial cells in the process of the role,and to detect proteasome activity to detect whether ICA ubiquitin-proteasome system influential.By WB were identified by the expression of ubiquitin immunofluorescence detection of chemical proteasome and the expression of UCH-L1.Results:ES cells were adherent colony-shaped growth.Undifferentiated ES cell clones nest or island-like shape;colony edge of a clear,smooth,and the feeder layer boundaries clear,no signs of differentiation;ES cells,closely arranged,ill-defined between cells.The hanging drop culture of ES cell aggregates,namely,EB,a three-dimensional spherical.Will be paved with gelatin EB transferred to the 24-well plate in culture,adherent continued after differentiation can be observed in myocardial cells appear.Myocardial cells similar to the spherical shape,with automatic rhythmic contraction center.Cardiac-specific sarcomeric protein actinin(α-actinin) and cardiac troponin T (troponin T) by immunofluorescence staining showed positive expression. Two-dimensional electrophoresis,different ICA differentiated phase induction and spontaneous differentiation of cardiac cell protein expression patterns varied significantly.In accordance with ES,EB,spontaneous differentiation d 5 +3,d 5 +7 myocardial cells,ICA-induced differentiation of d 5 +3,d 5 +7 order of myocardial cells,protein expression showed an overall upward trend,52 were identified by mass spectrometry protein spots,which increases protein spots 40,down protein spots 7,first downward and then upward protein spots 4.Differential expression of 52.In the embryonic body of the proteasome inhibitor Epoxomicin adding,after the beating of myocardial cells significantly reduced the number of shows that the proteasome in the Directional differentiation of ES cells into cardiomyocytes in the process played an important role.Determination of proteasome activity in the experiment,RA and ICA groups proteasome activity is higher than DMSO group,RA + E(inhibitors) and ICA+E group of proteasome activity is higher than E group. Ubiquitin protein expression and proteasome activity is consistent.Conclusion1.Through two-dimensional electrophoresis technology we build a targeted ES cells to differentiate into cardiac cells,protein expression patterns,ICA-induced and spontaneous differentiation of cardiac cell protein expression profiles varied significantly.2,ICA induction may be related to UPS system activity.Ubiquitin-proteasome system in the targeted ES cells to differentiate into myocardial cells in the process played an important role.Proteasome activity blocked a direct result of differentiation rate has declined markedly.