Studies On The Establishment Of A Myoblast Cell Line Of Grass Carp Skeletal Muscle And The Effect Of Arginine On Its Proliferation And Differentiation

Grass carp(Ctenopharyngodon idellus),as an important freshwater economic cultured fish with large scale in China,has the advantages of wide distribution,high nutritional value,low breeding cost,mature artificial propagation technology,good taste and strong adaptability.However,with the large-scale development of intensive farming,there are many problems in the farming process,such as high farming density,poor water quality,high incidence of pests and diseases,and decreased resistance,which also leads to the decline of the quality of artificially cultured grass fish.Different muscle fiber subtypes have a certain correlation with high-quality meat of livestock and poultry,and skeletal muscle development can be regulated at an early stage through the expression of specific genes or the addition of nutrients,which can usually produce lasting effects.Therefore,this study established a myoblast cell line derived from grass carp skeletal muscle as an in vitro model to study the mechanism of nutrition strategy regulating the development of grass carp skeletal muscle.According to the origin of the cell line,it was named CIM,and the biological characteristics and myogenic characteristics of the CIM cell line were characterized and identified,and then the influence mechanism of the cell line in nutritional regulation was preliminarily explored.The results of the study are as follows:1.Establishment and characterization of myoblast cell line of grass carp skeletal muscle.Firstly,primary cells were separated from grass carp skeletal muscle tissue by the collagenase-trypsin method,and the most suitable medium DMEM was selected by evaluating the vitality and growth of primary cells in different media.Then,the myogenic cells in cell suspension were purified by differential adhesion method according to the adhesion rate of myogenic cells and non-myogenic cells.The survival rate of CIM cell line after cryopreservation for 1-6 months was 79.78-95.06%,and no bacteria,fungi and mycoplasma were found in the culture process.The growth curve of CIM cell line showed an obvious"S"shape,and the population doubling time(PDT)was about 27.24 hours.The chromosome pattern number of CIM cell line is 48,and 48chromosome karyotypes are mainly composed of 8 pairs of metacentric chromosomes and 16 pairs of submetacentric chromosomes,and the matching rate between the mitochondrial 12S r RNA sequence of grass carp skeletal myoblast cell line and the known mitochondrial sequence of grass carp(Gen Bank accession number AY897013.1)is 99%.These results show that CIM cell line has similar genetic background and normal biological function to grass carp.2.Identification of myogenic and immortalized cell lines of grass carp skeletal muscle myoblasts.At the gene level,key myogenic regulatory factors myod,pax7,myog,myf5,myh1,myh2,myh4 and myh7 are expressed in primary cells and CIM cell lines,but the relative expression levels of each gene are different,myod,a myoblast-specific marker gene,has the highest expression levels in primary cells and CIM cell lines,but the difference in expression level is greatest in CIM cell lines.At the protein level,myogenic-specific marker proteins MyoD,Desmin and CD34 were expressed in CIM cell line,while fibroblast-specific marker protein PDGFRαwas not expressed in CIM cell line.Therefore,from the specific myogenic marker genes and proteins,it is proved that the cell type of CIM cell line derived from grass carp skeletal muscle tissue is mainly myoblast.The myogenic index and fusion index of CIM cell line were 8.96%-9.42%and 3-24 respectively after 10 d induction with 2%horse serum induction medium.The transfection efficiency of p EGFP-N1 was 23%to 24%after being successfully transferred into CIM cell line for 48 hours by liposome transfection.The telomerase activity of 5.06×10~5 cells in CIM cell line was 0.03 IU/mgprot by fish telomerase detection kit,so the CIM myoblast cell line established in this study is an immortal cell line with myotube fusion ability.3.The effect of arginine on the proliferation and differentiation of myoblasts of grass carp skeletal muscle.To make a preliminary application of this cell line,this study explored the effect of arginine on the proliferation and differentiation of grass carp skeletal muscle myoblasts using CIM cell line as a model.Different arginine concentrations have different effects on the growth and vitality of grass carp skeletal muscle cells.The effect of an arginine concentration of 100μg/m L on the growth and vitality of grass carp skeletal muscle cells is higher than other concentrations,and it can promote the growth and vitality of cells.This study also explored the effects of different growth times and arginine concentrations on myoblasts of grass carp skeletal muscle.The growth time,arginine concentration and the interaction between growth time and arginine concentration all have significant effects on the expression of key myogenic regulatory factors pax7,myod,myog,myh1,myh2,myh4 and myh7 in myoblasts of grass carp skeletal muscle,but have no significant effects on the enzyme activities of lactate dehydrogenase,malate dehydrogenase and succinate dehydrogenase.Treatment of CIM cell line with 100μg/m L arginine can significantly promote the relative expression of myogenic regulatory genes of myh7,igf-ⅱ,myod,myog,mrf4,myf5 and cyclind1,but has no significant effect on the relative expression of genes of myh1,myh2,myh4,fgf6and mstn.It is speculated that arginine may affect the proliferation of myoblasts in grass carp skeletal muscle by activating the transcription of igf-ⅱ,myod,mrf4,myf5 and cyclind1 genes.The results of western blot and immunofluorescence showed that arginine treatment significantly decreased the expression of myosin heavy chain subtypes Myh4 and Myh1,but significantly increased the expression of myosin heavy chain subtype Myh7.Therefore,arginine may affect the proliferation and differentiation of myoblasts of grass carp skeletal muscle by activating the transcription of myh7,igf-ⅱ,myod,mrf4,myf5,cyclind1 genes and the expression of myosin heavy chain subtype Myh7.In this study,the myoblast cell line of grass carp skeletal muscle was successfully established and the effect of arginine on the proliferation and differentiation of this cell line was preliminarily explored.This cell line can provide an in vitro model for studying the related mechanism of grass carp muscle development,and the study on the influence of arginine on this cell line can provide reference data and theoretical basis for the research on nutrition regulation of muscle development.