Effect Of Regulating PPARγ On Adipogenic Transdifferentiation Of Skeletal Muscle Satellite Cells In Yanbian Cattle

In recent years,with the continuous improvement of people’s living standards and the rapid development of animal husbandry,while pursuing the quantity of livestock products,consumers’ standards for the quality of meat products have become higher and higher than before.The most important factor in meat quality assessment is The index is the marble pattern,that is,the deposition of intramuscular fat(IMF).The content of IMF is highly related to the palatability of beef,which can affect the juicyness,tenderness and flavor of beef.Therefore,we must improve our understanding of the molecular mechanisms that affect IMF deposition so that genomic selection can be used to produce high-quality beef of consistent quality.The formation of intramuscular fat is partly due to the differentiation of adipocytes and partly due to the transdifferentiation of other muscle-derived stem cells.In this experiment,an animal model of Yanbian Cattle was used to regulate the expression of PPARy at the cell level by chemical induction to regulate the formation of fat.It is mainly divided into the following three experiments:Experiment 1:Establishment of Yanbian Cattle Skeletal Muscle Satellite Cell ModelIn order to investigate the effect of PPARy gene on intramuscular fat deposition in Yanbian Cattle,this study used Yanbian Cattle skeletal muscle semi-membrane muscle as a test material,and isolated cells using streptomycin digestion and isolation to establish Yanbian Cattle primary skeletal muscle satellite cells in vitro.Cultivation mode;Purification of cells by differential attachment method;Identification of skeletal muscle satellite cells by morphology and immunofluorescence;Detection and drawing of growth curves by MTS method to investigate the proliferation and differentiation of skeletal muscle satellite cells.The results of the study are as follows:The purified cells grew in a spindle shape,the cells contacted with each other,and the cells fused with each other to form multinucleated myotubes.After immunofluorescence staining,MyoD protein,Pax7 protein,and Desmin protein were positive,and MTS Cell proliferation was detected by the method and the curve was drawn as "S" type growth curve.Thus,the cells extracted from this experiment were identified as skeletal muscle satellite cells;and Yanbian Cattle skeletal muscle satellite cell models were successfully established for subsequent research.Experiment 2:Effect of ciglitazone on adipogenic and transdifferentiation of skeletal muscle satellite cells of Yanbian CattleThe purpose of this study was to investigate the effects of different concentrations of ciglitazone(CL:5 μM,CM:10 μM,CH:20 μM)on the adipogenic transdifferentiation of Yanbian Cattle skeletal muscle satellite cells.The results showed that the skeletal muscle satellite cells of Yanbian Cattle were treated with ciglitazone After 96 h,the satellite cells of Yanbian Cattle skeletal muscle changed from shuttle shape to round shape.Compared with the control group(CON),the myotubes of ciglitazone groups(CL,CM,CH group)decreased or even disappeared,and there were lipid droplets.The area of lipid droplets was highly related to the different concentrations of ciglitazone,and there was a dose dependent relationship(P<0.01).Compared with the control group(CON),ciglitazone was added The accumulation of triglycerides in each experimental groups(CL,CM,CH group)increased significantly(P<0.01),and there was a dose-dependent relationship between them.The real-time fluorescence quantitative RT-PCR results showed that:compared with the control group(CON),each experimental groups(CL,CM,CH group),PPARy,C/EBP α,C/EBP β,FABP4,FAS,ACC,SREBP1,LPL,GPR43,SCD,AMPKa,PLIN2,Pax3 and Pax7 gene expression increased significantly(P<0.01),and decreased the expression of CPT1,MyoD,MyoG,MYF5 and MRF4 significantly(Compared with the control group(CON),the expression of PPARγ,C/EBPα,SREBP1,PLIN2 and MyoD decreased significantly.Compared with the control group(CON),the content of adiponectin(ADP)in each experimental groups(CL,CM,CH group)was significantly increased(P<0.01).These results showed that ciglitazone could promote the differentiation of skeletal muscle satellite cells into adipocytes.Experiment 3:Regulation of PPAR y expression on Lipogenic and transdifferentiation of Yanbian Cattle skeletal muscle satellite cellsThe purpose of this study was to investigate the effects of ciglitazone(C:10 μM),GW9662(G:10μM)and their co addition(CG:10 μM ciglitazone+10 μM GW9662)on the adipogenesis and transdifferentiation of skeletal muscle satellite cells.Differentiation 96 h The results showed that compared with the control group(CON)and GW9662 group(G group),the myotubes of the group(C group)and the group(CG group)with ciglitazone alone(CG group)were decreased,the cells changed from spindle type to round,and lipid droplets were generated;compared with the control group(CON),the triglycerides of the group(C group)with ciglitazone alone(P<0.01)were significantly increased(P<0.01)Compared with the control group,PPAR γ,C/EBP α,C/EBPβSREBP1,FAS,ACC,LPL,FABP4 and GPR43 genes in group C and CG were significantly up-regulated,and the difference between the two groups was significant(P<0.01);Compared with the con group,the SCD gene in group C was significantly up-regulated,and there was no significant difference between the other two groups(P<0.01).Compared with the control group,the Pax3 gene in group C was significantly up-regulated(P<0.01),and the Pax7 gene was significantly down regulated(P<0.01);the MYOG,MyoD,MRF4,and MYF5 gene expression in group C and CG were significantly down regulated,and the MYF5 gene expression in group G was significantly up-regulated(P<0.01).The results of Western blotting showed that the expression of PPARγ,C/EBP α,PLIN2 and SREBP1 protein in group C and CG was increased,while that in group G was decreased,MYOG protein in group C was decreased,MyoD protein in group C was decreased,and that in group G was increased,compared with the control group,Pax7 protein in group C was increased The amount of protein expression decreased.Compared with the control group(CON),the content of adiponectin(ADP)in group C and CG increased significantly,while that in group G decreased significantly(P<0.01).These results indicate that up regulation of PPAR γ can promote the differentiation of skeletal muscle satellite cells into adipocytes,down regulation of PPAR y can inhibit the differentiation of skeletal muscle satellite cells into adipocytes.In conclusion,this study showed that the PPARy agonist ciglitazone could promote the differentiation of Yanbian Cattle skeletal muscle satellite cells into adipocytes.