The Regulation Of Mybph Gene In Myoblast Proliferation And Differentiation

Muscle development is a very complicated process,and regulation by many genes and transcription factors in the process of its development. Although it has identified a number of regulatory factors have a regulating effect on muscle development, but there are still a lot of genes or transcription factors of muscular development need to be researched.The report suggests that the expression of Mybph gene is differentiation in the process of muscle development,and it can regulate muscle development with other transcription factors.Therefore,in order to validate regulation function of Mybph gene for development of muscle cells,firstly,the expression level of Mybph gene in different organization of pig were detected; then predicted the location of Mybph protein via bioinformatics,and validation using laser confocal technique.Next,we detected the time-sequential expression of Mybph during myoblast differentiation.The last,we evaluated the effect of Mybph gene on myoblast proliferation,differentiation by constructing overexpression and interference vector of Mybph to treat myoblast,and using Real Time Cellular Analysis,immunofluorescence technique,western blot,flow cytometry and Real-time PCR.The specific results are as follows:1. The expression level of Mybph gene in heart,liver,spleen,lung,kidney,longissimus dorsi,ovary,endometrium,hypophsis,hypothalamus were detected with RT-PCR.Mybph gene was found expressed in various organizations,and expression high in longissimus dorsi.2. The results From bioinformatics analysis,the Mybph protein were localization in the cytoplasm.In addition, the Mybph protein was lcated in cytoplasm for using laser confocal technique,and the results in conformity with bioinformatics prediction.3. In order to research the influence of Mybph for muscle cell differentiation,the RT-PCR were used to determined the expression of the genes about muscle differentiation,such as genes of Myog,Myhc and Myomaker. The result said that up-expression of Mybph gene make the down-expression of these genes of muscle differentiation,and down-expression of Mybph gene make the up-expression of these genes of muscle differentiation.4. To investigate the role of Mybph in myoblasts differentiation,we assayed the mRNA expression level of Mybph gene in four different differential stage of C2C12 cells with RT-PCR technology.The result show that with cells differentiation,higher of expression.5. After Mybph gene expression was raised/lowered,the Real Time Cellular Analysis was used to detect the influence of proliferation of C2C12 cells.The results show that up-regulation of Mybph can inhibit cells proliferation,and down-regulated Mybph gene can promote cells proliferation.6. To detect the effect of raised/lowered Mybph gene on expression of Ki67,we carried out the immunofluorescence experiments and western blot.The results display that the gene of Mybph plays a promoting role in cell proliferation.7. The effect of raised/lowered Mybph gene on the cell cycle was detect with the folw cytometry,the result suggest that the gene of Mybph involved in cell cycle regulation,and the up-expression of Mybph can make more cells in G1 phase,down-expression of Mybph can make more cells in G2 and S phase.