Culture Of Wuzhumuqin Sheep's SMSCs And It's Expression Of Myogenic And Intramuscular Fat Related Genes During Differentiation

Wuzhumuqin sheep is an excellent fatstock in China with many advanteges such as high ingesting ability,genetic stability,high quality and high survival rate of lamb and high meat production.So its skeletal muscle satellite cells'culture model in vitro is particularly urgent and important.Skelatal muscle satellite cells?SMSCs?are adult stem cells that function as myogenic precursors for skeletal muscle growth and repair.This study based on SMSCs,we separated and purificated SMSCs from Wuzhumuqin sheep,and authenticated and studied their growth characteristics,then induced them into adipocytes and skeletal muscle cells,and analysed expression rules of muscle and intramuscular fat related genes during differentiation.The results showed that:We separated and purificated primary SMSCs from Wuzhumuqinsheep by modified primary explants culturing and different adherencing method,and then established continuously and stable Wuzhumuqinsheep SMSCs line.When cultured to the 18^thh generation,the cells still maintain a good cell morphology.Authenticating by immuno-histochemical staining and RT-PCR,in isolated cells,five protein markers--Desmin,?-Actinin,MYH1,Myostatin and Pax7 were showed immunoreactive product?red?.Meanwhile,positive pcr products were shown when ampilificated by specific primers Pax7,Myf5,MyoG,MSTN and MyoD.The immuno-histochemical and RT-PCR result both showed these cell line proved to be Wuzhumuqin sheep's SMSCs.Cell growth curve and survival rate proved that the obtained SMSCs from Wuzhumuqin sheep entered in logarithmic growth phase after second days of culture.In the culture cycle,cell viability was basically stable,but after eighth days of culture,the survival rate began to decrease slightly,Therefore,under the premise that cell confluence meets the requirements,the culture cycle should be 7 days.The cryopreservation solution prepared by this cell cryopreservation is 40%DMEM/F12+50%?FBS,Fetal bovine serum?+10%DMSO.After one-month preservation,the cell survival rate began to decrease significantly.The results of HE staining showed that the isolation and purification method of the SMSCs in the study was suitable for the establishment of a stable cell line,and the cells obtained in this study could be transmitted to 18 generations.After the SMSCs myogenic induction,a numerous myotubes shown on the 12^th day.In the myogenic induced cells,the relative expression of four factors of MRFs-Myf5,Myf6,MyoD and MyoG all increased,and compared with the expression level before induction,most of them showed significant difference?p<0.05?.The change of Myostatin gene expression is opposite to the four genes above,it decreased significantly,and showed extremely significant difference?p<0.01?.This phenomenon proves that the effect of Myostatin on skeletal muscle growth is largely inhibited after myogenic induction,it promotes the growth and development of skeletal muscle.SMSCs after adipogenesis induction on the 4^th day,a few round lipid droplets appeared around the cell.On the 8^th day after differentiation,a large number of round lipid droplets appeared around the cells.They showed positive?red?after Oil red-O staining,which proved that they were induced lipid droplets.On the 15^th day of differentiation,larger lipid droplets appeared,and Oil red-O staining result showed positive.The expression of FAS gene was not significant?p>0.05?during adipogenesis induction.But the expression of PPAR?gene was increased the difference was significant?p<0.05?.The differentiation results of skeletal muscle satellite cells at different ages are as follows:for embryonic skeletal muscle satellite cells,the expression of four genes in the MRFs family-Myf5,Myf6,MyoD and MyoG,increased during myogenic induction,and decreased during adipogenesis induction.The Myostatin gene behaved approximately the same in different ages of individuals,both showed downregulation during myogenic and adipogenic differentiation.The expression of intramuscular fat related genes FAS and PPAR?appeared regularly in the individuals of different months,they decreased in the process of myogenic differentiation,and significantly increased in the process of adipogenesis induction?p<0.05?.Establishing a highly efficient and stable SMSCs separation,purification and identification system for Wuzhumuqin sheep,analyse the expressing rule of MRFs gene and MSTN gene during cell deferation and sheep's prenatal to postnatal development will promote the growth and hypertrophy of skeletal muscle and provide good scientific theory data to improve its meat production and meat quality.