Follistatin Effect On Proliferation Of Duck Skeletal Muscle Satellite Cell And The Preliminary Research Of The Mechanism

Follistatin as member of TGF-? super-family,skeletal muscle of embryo stage and the process of growth and development play a wide range physiological role in mammalian,in skeletal muscle Proliferation,regulating liver,regeneration of red blood cells,and nerve cells differentiation,etc.a variety of signal regulating circuit also play an important role.Therefore,explore Follistatin effects on proliferation of duck skeletal muscle satellite cell,at the same time hope to explore Follistatin to the mechanism of action of duck skeletal muscle satellite cell proliferation,hope for further research on Follistatin regulation of duck skeletal muscle growth and development mechanism lay the foundation?To hatch 14d duck embryo as test material,using differential adherence method isolate skeletal muscle satellite cells,using the concentration 0,1,10,100 ng/ml to deal with duck skeletal muscle satellite cells,respectively.After incubation 36h,the proliferation of skeletal muscle satellite cells are detected by CCK-8.The cells were stained with pax7 Antibody by immunofluorescence staining and DAPI nuclear staining.Using flow cytometry instrument detection changes of skeletal muscle satellite cell cycle.Real-time qPCR was performed to measure the alterations in the expression of MyoD,smad2/3,TGF-? and myogenic proliferation marker genes caused by Follistatin supplement.Western blot detected Smad2 and Smad3 protein expression and its phosphorylation level.Determine Follistatin effect on proliferation of skeletal muscle satellite cell and its mechanism of action.The main results are as follows:1.By using Pax7 antibodies immunofluorescence staining,the results show that more than 95%of Pax7 positive expression and description of skeletal muscle satellite cells cultured cells.2.Detected different concentration Follistatin(0,1,10,100 ng/ml)influence on skeletal muscle satellite cells proliferation by CCK 8,the results showed that 10 ng/ml of Follistatin most obvious effect on skeletal muscle satellite cell proliferation(P<0.01).3.Pax7 is adopted to the immunefluorspar colored dye showed that Pax7 positive expression was significantly increased(P<0.05).Flow cytometry detection changes of skeletal muscle satellite cell cycle,compared with control group,the results of G1 phase,the cells decreased significantly(P<0.05),while a significant rise in G2 phase and S phase(P<0.05).Real-time qPCR method to detect the expression of PCNA and MyoD gene,Compared with the control group,the results showed a significant rise in gene expression of PCNA(P<0.01),significantly reduce the amount of MyoD gene expression(P<0.05).Through the use of real-time qPCR method to detect the expression of TGF-beta.Smad2 and Smad3 gene,Compared with the control group,the results showed the expression of TGF-beta.Smad2 and Smad3 gene quantity were significantly higher(P<0.01).Western blot test results show that Smad2 and Smad3 protein expression and its phosphorylation level significantly increased(P<0.05).4.After using LY2109761 inhibitor treatment duck skeletal muscle satellite cells,Using flow cytometry instrument to detect the change of skeletal muscle satellite cell cycle,the results show that compared with the control group,LY(LY2109761)inhibitors in the treatment group,the marked increase in G1 phase(P<0.05),S phase cells and G2 stage significantly reduced(P<0.05).Realtime qPCR detection gene expression of MyoD,PCNA,TGF-beta,Smad2 and Smad3,Results found that MyoD gene and PCNA were present the rise and fall significantly(P<0.05),TGF-beta gene expression quantity did not change significantly,Smad2 and Smad3 gene expression quantity significantly reduced(P<0.05),LY2109761 inhibitors and Follistatin co-treatment duck skeletal muscle satellite cell,the marked increase in G1 phase(P<0.05),S phase cells and G2 stage significantly reduced(P<0.05).MyoD gene and PCNA can present the rise and fall significantly(P<0.05),TGF-beta gene expression were significantly higher(P<0.05),Smad2 and Smad3 gene expression quantity significantly reduced(P<0.05),Western blot detection Smad2 and Smad3 protein expression and phosphorylation levels,the results show that Smad2 and Smad3 protein expression and phosphorylation levels decreased significantly(P<0.05).