Effect Of Inhibitor Of Differentiation-2 On Myoblast Cell Proliferation And Apoptosis

Skeletal muscle regeneration is a process in which myoblasts were induced to proliferation and differentiation.The process contains intra-cellular gene regulation, polykaryocyte's generation,formation of sarcotubule and formation of full-grown fibrocyte via differentiating,and then regeneration of the new skeletal muscle.To induce myoblasts proliferation and differentiation and inhibit myoblasts apoptosising are the important pathway of skeletal muscle regeneration.Inhibitor of differentiation/DNA binding-2(Id2)belongs to the Id proteinic family of Helix-loop-Helix(HLH)proteinic family.It has the identical ability of forming dimer with bHLH.Nevertheless,because of it defects the basic amino acid sequence essential for binding DNA,after Id2 binded with bHLH to form dimmer(Id-bHLH),it inhibits bHLH binding with DNA and other bHLH transcription factor with tissue specificity.So it down regulate the transcribe activity of Myogenic regulatory factors, thereby inhibiting myoblast differentiation.It's already been evidenced that Id2 could promote cell proliferation and inhibit cell differentiation in majority cell lines.But the research about effect on cell apoptosis of Id2 is less.Some research indicated that Id2 also played an important role in cell apoptosis of many cell lines.However,existing research does not solve the rigorous mechanism of action of Id2 yet.For example,how did Id2 regulate punctually MyoD,Myogenin and MRF4 of MRFs family? Whether the high expression of Id2 are correlated with Myogenin's low expression? The research should be continued to study the pathway regulated skeletal muscle loss related to apoptosis by Id2,and the relation between Id2 and skeletal muscle apoptosis.Whether Id2 could regulate the cell proliferation,apoptosis and differentiation of skeletal muscle myoblast or not still to solve.We established the C₂C₁₂myoblast cell line in vitro culture as us experimental model,using exogenous reactive oxygen species to stimulate C₂C₁₂to create oxidative stress for inducing cell proliferation and apoptosis,in order to find the optimal effect condition that induced C_2C12cell proliferation and apoptosis.We constructes the pEGFP-C2-Id2 eukaryotic expression vector that have exogenous Id2 gene,and transferred the vector into C₂C₁₂cell by the electroporation.So we will investigate the effect of Id2 on C₂C₁₂cell proliferation and apoptosis,thus to provide experimental reference for researching the relation between Id2 and skeletal muscle.[Objective]To explore the effect of Id2 in C₂C₁₂cell line during proliferation,differentiation and apoptosis.[Methods]1.RT-PCR method was used to amplify the entire Id2 cDNA.The pGEM-T and Id2 cDNA were ligated by T₄DNA ligase.The cloning vectors and the pEGFP-C2(eukaryotic expression vector)were first cut by EcoR I,and then ligated with Id2 cDNA by T₄DNA ligase again.The enzyme analysis,PCR identify and DNA sequencing methods were used to confirm the recombined vector.2,Hydrogen peroxide induced C₂C₁₂cell proliferation and apoptosis.MTT and Hoechst33342/PI double staining methods were used to optimize the effective condition.With this method,we examed the Id2 expression in C₂C₁₂cell during proliferation and apoptosis used by immunity fluorescence.3,Hydrogen peroxide,LPS and horse serum were used to induced C₂C₁₂cell proliferation,apoptosis and differentiation,The Id2 expression in C₂C₁₂cell were observed.[Results]1.We successfully constructed pEGFP-C2-Id2 eukaryotic expression vector and transfected it into C₂C₁₂cells and had highly transfectional efficiency.2.The optimal effective condition that induced C₂C₁₂cell proliferation and apoptosis by hydrogen peroxide are respectively as follow:25μmol/L,48h;50μmol/L,48h.Id2 expression showed in both groups.,proliferative and apoptotic C₂C₁₂cells:compared to normal C₂C₁₂cells,25μmol/L group was the most powerful;50μmol/L group was the next,Id2 expression was attenuated along with H₂O₂ concentration increased.3,C₂C₁₂cells were treated by H₂O₂,LPS and horse serum separately after 48h,Id2 expression all appeared proliferative and apoptotic C₂C₁₂cells:compared to normal C2C12 cells,the cell treated with 2%horse serum had no Id2 expression;25μmol/L H₂O₂ group was the most powerful;25μmol/L H₂O₂ had massive Id2 expression;Id2 expression was attenuated along with LPS concentration increased in LPS treatment group.[Conclusion]1.Low concentration(25μmol/L)of H₂O₂ promotes C₂C₁₂cell proliferation and Id2 expression,High concentration(50μmol/L or higher)of H₂O₂ prormotes C₂C₁₂cell apoptosis and Id2 expression.Oxidative stress can promote or induce Id2 expression.2.Id2 regulates C₂C₁₂cell growth:Id2 could not only promote or induce C₂C₁₂cell proliferation but also promote or induce C₂C₁₂cell apoptosis;Id2 inhibit C₂C₁₂cell differentiation.