| With the improvement of living standards,the proportion of meat in diet composition is increasing gradually.People have higher requirements on the quality of meat food.At present,there are a wide variety of beef cattle with low yield and uneven quality in our country.The yield and quality of the beef can not to meet people's demand.The way to solve this problem most rapidly and effectively is to obtain high-quality beef cattle through genetic breeding.Furthermore,latent skeletal muscle satellite cells are activated after skeletal muscle injured,to repair the damaged muscles through regenerate muscle fibers through proliferation and differentiation.So,the study on muscle differentiation not only has great commercial value,but also can provide novel ideas for the treatment of related diseases in medical field.Ran is the most abundant small G protein in eukaryotic cells.It takes part in nucleocytoplasmic transport,DNA replication and RNA transcription in mitotic interphase cells.And participates in the process of nuclear membrane rupture,spindle assembly,chromosome distribution,nuclear membrane assembly and cell division during mitosis or meiosis phase.Muscle differentiation is the process of myogenic cells form muscle fibers with contractile functions through proliferate,differentiate and fuse with each other into polynuclear myotubes.In this study,mouse C2C12 myoblasts was used as the research object.the Ran overexpression plasmids and Ran si RNA interference fragments were used to study the effect of Ran on C2C12 myoblasts differentiation.And the possible mechanisms were explored then.The main research contents and results obtained are as follows:(1)The expression of Ran during C2C12 myoblasts differentiation: The protein samples were collected on the 0 day to 5 day of differentiation C2C12 myoblasts for Western blot analysis.The results showed that,with the differentiation of C2C12 myoblasts,the protein expression level of Ran were increased gradually.(2)The distribution of Ran during C2C12 myoblasts differentiation: Indirect immunofluorescence staining undifferentiation stage,early differentiation stag e,middle differentiation stage and late differentiation stage C2C12 myoblasts,it was found that Ran accumulated in nucleus of C2C12 myoblasts at undifferentiation stage.And with the increase of differentiated time,the concentration of Ran increased in the cytoplasm.(3)The effect of Ran overexpression on C2C12 myoblasts differentiation: Overexpression plasmids of Ran were transfected into C2C12 myoblasts.Western blot analysis showed that,the protein expression level of Ran was significantly increased and the protein expression level of Myo G and MYH was significantly increased also;the results of indirect immunofluorescence stain indicated that,the myotube fusion rate of C2C12 myoblasts was significantly increased.The increased protein expression levels of Ran promotes C2C12 myoblasts differentiation.(4)The effect of si RNA interference of Ran on C2C12 myoblasts differentiation: The si RNA interference fragments of Ran were transfected into C2C12 myoblasts.Western blot analysis showed that,the protein expression level of Ran was decreased and the protein expression level of Myo G and MYH was decreased also;the results of indirect immunofluorescence stain indicated that,the myotube fusion rate of C2C12 myoblasts was significantly decreased.The decreased protein expression level of Ran inhibits C2C12 myoblasts differentiation.(5)The effect of TGF-?/Smad signaling pathway on C2C12 myoblasts differentiation: Inhibitor LY2109761 of TGF-?/Smad signaling pathway was added to the C2C12 myoblasts differentiation culture system.Western blot analysis showed that,the phosphorylation level of Smad2 was decreased,the protein expression level of Myo G and MYH was significantly decreased,the differentiation of C2C12 myoblasts was suppressed.(6)The effect of Ran on TGF-?/Smad signaling pathway: Using overexpression plasmids and si RNA interference fragments of Ran to increase or decrease the protein expression of Ran during C2C12 myoblasts differentiation.Western blot analysis showed that,at 2 d ay of C2C12 myoblasts differentiation,the protein expression level of Smad2 was significantly increase d with increased expression of Ran and significantly decreased with decreased expression of Ran.But the protein expression level of Smad2 was not affected by Ran expre ssion at 4 day and 6 day of C2C12 myoblasts differentiation.From 2d to 6d of C2C12 myob lasts differentiation,Phosphorylation of Smad2 was significantly increased with increased expression of Ran and significantly decreased with decreased expression of Ran.The increased Ran expression activates the TGF-?/Smad signaling pathway and the decreased Ran expression inhibits the TGF-?/Smad signaling pathway.(7)The relationship between TGF-?/Smad signaling pathway and Ran in the differentiation of C2C12 myoblasts: After LY2109761 treatment,the phosphorylation level of Smad2 was decreased,the protein expression level of Myo G and MYH was significantly decreased.LY2109761 and Ran overexpression plasmids were added to the C2C12 myoblasts differentiation culture system in the meantime.The phosphorylation level of Smad2 and the protein expression levels of Myo G and MYH were increased to the level of differentiated cells without LY2109761.The results showed that Ran promotes C2C12 myoblasts differentiation by increasin g the protein expression level and phosphorylation level of Smad2 in the TGF-?/Smad signaling pathway. |
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