滩羊骨骼肌卫星细胞体外培养及成肌和成脂诱导分化研究

In vitro Culture of Skeletal Muscle Satellite Cells of Tan Sheep and Induction of Myogenesis and Adipogenesis

为了建立滩羊骨骼肌卫星细胞(Skelatal muscle satellite cells,SMSCs)的体外培养体系,并探讨在成肌和成脂诱导分化过程细胞形态、标志基因表达等变化规律,该试验采集10日龄健康滩羊(公羔)腿部肌肉组织,用I型胶原酶和胰蛋白酶消化分离骨骼肌卫星细胞进行原代和传代培养,并进行成肌和成脂诱导分化。采用qRT-PCR检测成肌分化基因(Pax7、Desmin、MyoG)及成脂分化关键基因(PPARγ、C/EBPα、FAS)mRNA在诱导前、后的表达水平变化。结果表明:(1)SMSCs在酶消化法分离后约9 h后开始贴壁,呈现为小梭形与不规则三角形。经传代后,细胞呈现梭形及不规则三角形,可见部分融合细胞,折射率增加;培养过程中发生更多融合,细胞长梭形且平行排列紧密,呈长管状;(2)细胞接种后的潜伏期为0~4 d,此时完成贴壁和伸展;4~6 d进入对数生长期,增殖迅速;第6天开始进入到平台期,细胞增殖缓慢;(3)SMSCs成肌诱导分化第8天,明显观察到细胞核和肌管;成脂诱导第8天时细胞内沉积大量脂肪滴,油红O着色呈明显红色;(4)SMSCs成肌诱导第8天Pax7表达量显著降低(P<0.05),为诱导第0天的0.08倍;MyoG和Desmin在诱导第8天表达量显著增加(P<0.05),分别是诱导第0天的11.03倍和5.48倍。SMSCs成脂诱导分化第8天与诱导第0天相比,PPARγ表达量增加,差异不显著(P>0.05);而C/EBPα和FAS的表达量显著增加(P<0.05),表达水平分别提高5.51倍和2.01倍。该研究成功分离滩羊骨骼肌卫星细胞,并具有较强成肌和成脂分化能力,为滩羊骨骼肌的发育及再生机制提供了可靠的细胞模型。

The purpose of this study was to establish a culture system of Tan Sheep Skeletal Muscle Satellite Cells(SMSCs)in vitro,and to explore its characteristics of adipogenesis and myogenesis during differentiation induction.Leg muscle tissues of ten-day-old Tan sheep were collected,digested with type I collagenase and trypsin to isolate skeletal muscle satellite cells,and the cells were primary and subcultured,and morphological observations were made at the same time.The obtained cells were subjected to myogenic and adipogenic induction,respectively,and identified by immunofluorescence and oil red O staining.At the same time,qRT-PCR was used to detect the expression levels of Pax7,Desmin,MyoG,PPARγ,C/EBPα,FAS mRNA,the key genes of myogenic and adipogenic differentiation,before and after induction.Results showed(1)SMSCs began to adhere after about 9 h after separation,and the adherent cells appeared as fusiform and irregular triangles.After passage,the cells showed a fusiform and irregular triangle shape,showing that part of the cells were fused and the refractive index increased;during the cultivation process,more fusion occurred,and the cells were long fusiform and closely arranged in parallel,showing a long tube shape.(2)The incubation period after cell inoculation was 0~4 d.At this time,the adherence and extension of the cells were completed;4~6 d were the logarithmic growth phase,and the cells proliferated rapidly.After 6 d,they entered the plateau phase,and the cell proliferation was slow.(3)On the 8th day of myogenic induction differentiation of SMSCs,the expression of MHC was detected by cellular immunofluorescence.The results showed that the immunostained nuclei and myotubes could be clearly observed on the 8th day of differentiation;SMSCs deposited a large number of fat droplets in the cells on the 8th day of adipogenic induction,and oil red O was stained obviously red.(4)The expression of Pax7 on the 8th day of induction of SMSCs myogenesis was significantly reduced(P<0.05),which was 0.08 times that of the 0th day of induction;the expression of MyoG and Desmin on the 8th day of induction of myogenic induction of SMSCs was significantly increased,which was 11.03 times and 5.48 times of it on the 0th day of induction.Compared with the 0th day of induction,the expression of PPARγincreased on the 8th day of SMSCs adipogenic differentiation,and the difference was not significant;the expression of C/EBPαand FAS increased significantly;the expression of C/EBPαand FAS on the 8th day of SMSCs adipogenic induction,which was 5.51 times and 2.01 times of induction on day 0,respectively.It is concluded that this study successfully isolated Tan sheep skeletal muscle satellite cells which have strong myogenic and adipogenic differentiation capabilities.It provides a reliable cell model for the development and regeneration of Tan sheep skeletal muscle.