鸡骨骼肌卫星细胞的分离培养与鉴定

Isolation,Culture and Identification of Muscle Satellite Cells of Chicken

旨在建立鸡骨骼肌卫星细胞(muscle satellite cell,MSC)体外分离、培养、纯化、诱导分化及鉴定的方法。选取12胚龄SPF鸡胚,综合松散肌肉纤维和游离纤维基质层细胞等原理,对分离细胞使用的酶以及后续的细胞纯化、诱导分化方法进行优化;并从形态学、细胞分化能力、生长曲线、免疫荧光、RT-PCR等方面对MSC进行鉴定,从而提出一种鸡骨骼肌卫星细胞的混合酶消化分离培养方法。结果表明,刚分离纯化后的细胞折光性强,24 h贴壁展开后呈纺锤状;待诱导分化后能形成排列整齐的多核肌管;CCK-8测定细胞生长曲线呈典型的“S”型;优化后,细胞存活率为(90.82±1.294)%,细胞纯度为(90.44±1.264)%;免疫荧光检测标志性基因Pax7、Desmin表达阳性;RT-PCR检测标志性基因Pax7、MyHC、MyoD1呈阳性;并且分化前标志性基因Pax7表达量是分化后的1.705倍,分化后期标志性基因MyHC表达量是分化前的13.073倍。该试验建立了一种快速简便的鸡骨骼肌卫星细胞的体外培养方法,为研究鸡骨骼肌细胞生物学机制、肉鸡品种优化、细胞移植修复提供良好的细胞模型。

The purpose of the study was to establish methods for isolation,culture,purification,differentiation and identification of chicken skeletal muscle satellite cells(MSC)in vitro.Selecting SPF chicken embryos of 12 embryonic age,integrating the principles of loose muscle fiber and free fiber matrix layer cells,the enzymes used to separate cells,cell purification and differentiation induction methods were optimized,and the MSC was identified from the aspects of morphology,cell differentiation ability,growth curve,immunofluorescence detection,RT-PCR identification,etc.Therefore,a method of digesting,separating and culturing chicken skeletal muscle satellite cells with mixed enzymes was proposed.The results showed that the separated and purified cells had strong refractive index and adhered to the wall after 24 hours,showing a spindle shape;after induced differentiation,it could form neatly arranged multinucleated myotubes;the cell growth curve measured by using CCK-8 method showed a typical“S”shape;after the optimization,the cell survival rate was(90.82±1.294)%,the cell purity was(90.44±1.264)%;immunofluorescence detection of the marker genes Pax7 and Desmin were positive;RT-PCR detection of the marker genes Pax7,MyHC,MyoD1 were positive;and the expression of the marker gene Pax7 before differentiation was 1.705 times that after differentiation,and the expression of the marker gene MyHC after differentiation was 13.073 times that before differentiation.This study established a quick and simple method for culturing chicken skeletal muscle satellite cells in vitro,which provides a good cell model for studying the biological mechanism of chicken skeletal muscle cells,optimization of broiler breeds,and cell transplantation repair.