【作者】 乌日罕；

【导师】 格日勒图（BORJIGIN Gerelt）；

【作者基本信息】 内蒙古农业大学 ， 食品科学， 2018， 博士

【摘要】 乌珠穆沁羊是中国著名的优良肉畜品种,为了研究其肌肉生长发育机制,建立体外培养模型,并研究如何提高该肉畜的产肉性能与肉质为本研究主要目的。骨骼肌卫星细胞(SMSCs)主要与骨骼肌生长和修复相关,因此本研究主要以SMSCs为模型,进行分离纯化,鉴定与培养特性分析,并诱导其成为脂肪细胞与骨骼肌细胞,同时探讨了SMSCs在分化过程中肌肉与肌内脂肪相关基因的表达变化。研究结果表明:通过改良传统的细胞分离方法—原代外植块培养法,结合改良的差速贴壁法分离得到了高纯度的乌珠穆沁羊骨骼肌卫星细胞,此方法所得细胞培养至18代均未出现衰退现象,细胞仍然能够保持较好的形态特征。将所得卫星细胞通过免疫组织化学实验结合RT-PCR方法进行鉴定,证明所分离得到的细胞确实为骨骼肌卫星细胞。培养特性研究结果表明,所得SMSCs在细胞汇合度符合要求的前提下,培养周期不能超过7天;最佳冷冻保护液配方为40%DMEM/F12+50%胎牛血清+10%DMSO。HE染色结果证明,所建立乌珠穆沁羊骨骼肌卫星细胞分离纯化方法适于建立该细胞稳定细胞系,且可继代培养至18代。成肌诱导后细胞的形态学变化表明,成肌诱导后的第12天开始形成大量多核肌管。成肌诱导后的MRFs家族四个基因相对表达量均呈现上升趋势)。Myostatin基因表达量变化相反,在成肌诱导后呈现极显著性下降(p<0.01)。此现象证明在成肌诱导后,Myostatin抑制骨骼肌生长的作用受到了很大程度的抑制,从而促进了骨骼肌的生长发育。随着月龄的增加,MRFs家族四个因子表达量随之下降,Myostatin表达量则呈现上升趋势。成脂诱导后细胞的形态学变化表明,相较于成肌分化,骨骼肌卫星细胞的脂肪分化速度较慢,SMSCs在成脂诱导后第15天,出现较大的脂滴,Oil red-o染色呈阳性。成脂诱导后FAS基因表达量无明显变化,PPARγ表达量提高且差异显著(p<0.05)。随着月龄的增加,肌内脂肪相关基因的表达量随之增加。不同月龄个体骨骼肌卫星细胞的分化结果如下:胚胎期个体骨骼肌卫星细胞中的MRFs家族四个基因的表达量在成肌诱导时呈现不同程度的上调,成脂期间呈现不同程度下调。Myostatin基因在不同月龄个体中的表达量大致相同,在成肌与成脂分化期间均有不同程度下调现象。肌内脂肪相关基因FAS和PPARγ在不同月龄个体中出现了规律性的变化,成肌分化中不同程度下调,成脂分化中不同程度上调。本研究结果在促进乌珠穆沁羊骨骼肌生长与肥大,从而促进瘦肉生产率并提高肉品质以及保护地区优势品种——乌珠穆沁羊,建立其体外培养机制等方面,均可以提供科学的理论数据。更多还原

【Abstract】 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,ɑ-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 PPARγgene 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 PPARγappeared 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.更多还原