| As a dynamic organelle in fat cells,lipid droplets were composed of a hydropho-bic neutral lipid core surrounded by a phospholipid monolayer and perilipid droplet proteins.As one of the main perilipid droplet proteins,PLIN2 acts as a barrier protectant for lipid droplets by inhibiting the contact of adipocyte TAG hydrolase with the surface of lipid droplets.Skeletal muscle satellite cells,as pluripotent stem cells,can be induced to differentiate into a variety of cells under different conditions.Perilipid droplet protein can control proper lipid storage in skeletal muscle,and PLIN2 is one of the most abundantly expressed perilipid droplet protein in skeletal muscle.Oleic acid(OA),as the most representative monounsaturated fatty acid in beef,plays an important role in regulating lipid metabolism and the formation of intramuscular marbling.In beef adipose tissue,the higher the oleic acid content,the higher the intramuscular fat content.The previous research of our laboratory found that oleic acid can promote the formation of lipid droplets in Yanbian bovine skeletal muscle satellite cells and pre-adipocytes,and significantly increase the expression level of PLIN2 gene.Therefore,PLIN2 gene is likely to be an important genetic marker gene for intramuscular lipid accumulation in beef cattle.Regulating the expression of PLIN2 gene may clarify the relevant mechanism of intramuscular fat accumulation.Based on this,this study used the established Yanbian bovine skeletal muscle satellite cells as a research model and added oleic acid to study its effect on the adipogenic differentiation of skeletal muscle satellite cells,in order to study the path of adipogenic differentiation of Yanbian bovine skeletal muscle satellite cells caused by oleic acid.The molecular regulation mechanism of PLIN2 provides a molecular theoretical basis,while using transcriptomics and proteomics technology to provide basic materials for further elucidating the molecular mechanism of the PLIN2 gene in the process of fat formation.The main research contents were as follows:1.Isolation,culture and differentiation of Yanbian cattle skeletal muscle satellite cellsThe satellite cells were isolated and identified from the longissimus dorsi muscle of 12 day old calves by streptavidin digestion and differential adhesion method.The results showed that the growth curve of skeletal muscle satellite cells conformed to the normal growth law;immunofluorescence staining showed that the specific marker genes Pax7,desmin and MyoD of 13skeletal muscle satellite cells showed green fluorescence,and MyoD and Pax7 were expressed at all stages of myogenic induction,indicating that the muscle satellite cells were in an activated state and differentiated into myoblasts after induction.These results indicated that Yanbian cattle skeletal muscle satellite cells were successfully isolated and obtained in this study,which can provide an in vitro cell model for the study of Yanbian cattle muscle differentiation and adipogenesis related regulation process.2.Analysis of Bioinformatics and Expression Law of Yanbian Cattle PLIN2 geneIn this study,18 month old ovariectomized Yanbian cattle were selected as the research object.The PLIN2 gene of Yanbian cattle was obtained,and its coding sequence was analyzed by bioinformatics to detect the expression level of different tissue parts and the expression pattern after myogenic differentiation.The results showed that the CDs region of PLIN2 gene of Yanbian cattle was cloned successfully,and the homology was the highest with that of cattle and buffalo.The encoded protein was an unstable mixed protein with ?-helix as the main protein,which was connected by irregular coil,had certain hydrophilicity,and had no signal peptide.The expression level of PLIN2 gene was the highest in small intestine,followed by muscle and kidney.After myogenic differentiation,the whole protein showed a downward trend.The results of this study can provide reference for further study on the function of PLIN2 gene.3.The effect of oleic acid on the adipogenic differentiation of Yanbian bovine skeletal muscle satellite cells and its transcriptomics analysisDifferent concentrations of oleic acid were added to induce adipogenesis and differentiation of skeletal muscle satellite cells in vitro.The results showed that:(1)After the addition of oleic acid,lipid droplets were formed in Yanbian bovine skeletal muscle satellite cells,and the amount of lipid droplet formation,accumulation of triglycerides,and adiponectin content all increased significantly(P<0.05),showing an upward trend.(2)The measurement results of related factors showed that the myogenesis-related factors Pax3,MyoD,MRF4 and Myf5 were significantly down-regulated(P<0.05);the fat-related factors C/EBP?,C/EBP?,SREBP1 and PPAR? were significantly up-regulated(P<0.05);Fatty acid metabolism-related factor SCD was significantly down-regulated(P<0.05),and PLIN2 gene was significantly up-regulated(P<0.05).(3)A total of 13,951 single genes were obtained from RNA-Seq analysis,and there were a total of 278 differentially expressed genes among the treatment groups.GO enrichment analysis shows that differential genes are involved in a variety of biological processes;in molecular function,they are related to binding activity,transcription regulation activity,catalytic activity,etc.;in the category of cell components,most genes are enriched in cells and organelles,Film,etc.KEGG enrichment analysis showed that the main enrichment pathways of differentially expressed genes were AMPK signaling pathway,PPAR signaling pathway,fatty acid metabolism pathway,etc.(4)The PLIN2 gene exists in the PPAR signaling pathway,and its expression is regulated by PPAR?.It has a major molecular function relationship with MMP1,SORBS1,and CD36,and plays an important role in the process of adipogenesis.4.The effect of interfering PLIN2 gene on the adipogenic differentiation of Yanbian bovine skeletal muscle satellite cells induced by oleic acid and its proteomic analysisThe purpose of this experiment was to find the protein function analysis of the adipogenic differentiation of Yanbian bovine skeletal muscle satellite cells by oleic acid after PLIN2 expression was inhibited.The results showed that:(1)Compared with the negative control group,the amount of lipid droplets and triglycerides produced after interference with PLIN2 was not significantly different(P>0.05),and adiponectin decreased significantly(P<0.05);(2)The measurement results of related factors showed that the myogenesis-related factors Pax7 and MyoD were significantly down-regulated(P<0.05),and Myf5 was unchanged(P>0.05);the fat-related factors PPAR? and C/EBP? genes were significantly up-regulated(P<0.05),C/EBP? gene was significantly down-regulated(P<0.05);fatty acid metabolism-related factors FABP4,SCD genes were significantly up-regulated(P<0.05),CD36,PLIN2 were significantly down-regulated(P<0.05),and CPT1 B genes were not significantly different(P>0.05).(3)Proteomics analysis found that 5392 detected proteins were retained after pretreatment;compared with the negative control group,there were 112 differentially expressed proteins,of which 62 were up-regulated and 50 were down-regulated;homologous protein clusters were concentrated in signal transduction Guidance mechanism,general function prediction,intracellular transport,secretion and vesicle transport,etc.;subcellular localization is mainly concentrated in the nucleus,cytoplasm,and extracellular.GO annotation enrichment analysis shows that the differential proteins are in the category of cell components,most of which were enriched in the cell,intracellular part,cytoplasm,etc.;in the molecular function category,most of the functions are combined with ions,small molecules,and drugs Binding and other related;involved in the negative regulation of cell response to growth factor stimulation,cell-matrix adhesion junction assembly,adhesive assembly and other biological processes.KEGG analysis showed that differentially expressed proteins are mainly enriched in metabolic pathways,thermogenesis pathways,and influenza A pathways.(4)There was a major correlation between PLIN2 protein and ACSL6 protein,ALB protein,LPCAT2 protein and SPART protein in terms of protein function.In conclusion,Yanbian cattle skeletal muscle satellite cells were successfully isolated in this experiment,and the bioinformatics and expression rules of PLIN2 gene were analyzed.Through the establishment of cell model,oleic acid promoted Yanbian cattle skeletal muscle satellite cells to differentiate into adipocytes.At the same time,the interaction between PLIN2 gene and adipocytes was explained by transcriptomics and proteomics,This study provides a theoretical basis for the adipogenic differentiation of Yanbian cattle skeletal muscle satellite cells induced by oleic acid,and provides a basic material for the regulation of intramuscular fat deposition by molecular nutrition. |
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