绵羊骨骼肌卫星细胞的体外培养和诱导分化

发布时间：2018-04-15 05:36

本文选题：绵羊 + 分离培养　；参考：《内蒙古大学》2012年硕士论文

【摘要】：骨骼肌卫星细胞是成体肌源性干细胞,在各种成体干细胞研究中越来越受到人们的关注。虽然从一些物种(如小鼠、大鼠和人)的骨骼肌中已经成功分离出卫星细胞,但对于家畜(如牛、绵羊)的研究却很少。在家畜转基因的研究和生产过程中,需要在动物生产之前对转基因细胞系进行基因表达的鉴定和验证。因此,家畜骨骼肌卫星细胞体外培养模型的建立就显得特别迫切和重要。本研究以绵羊骨骼肌卫星细胞作为研究对象,重点探讨卫星细胞的分离、纯化及体外培养方法,并对得到的细胞进行鉴定,旨在建立可稳定传代的绵羊卫星细胞系及其鉴定方法,为今后绵羊骨骼肌卫星细胞在家畜繁育和再生医学等相关方面的研究和应用提供实验依据。用两步酶消化法和差速贴壁法分离和纯化绵羊骨骼肌卫星细胞,并对其进行鉴定和诱导分化,分析其多能性。结果表明,1.1%Ⅰ型胶原酶和0.25%胰蛋白酶两步消化法分离绵羊骨骼肌卫星细胞效率较高,含20%FBS+10%马血清的培养基更有利于绵羊骨骼肌卫星细胞的体外培养。用免疫组化和流式细胞术检测绵羊骨骼肌卫星细胞中几个标记基因Desmin、α-SarcOmeric Actinin、MyOD1、Myf5和PAX7的表达情况,本研究得到的细胞中这5个基因表达均呈阳性,说明分离得到的细胞为绵羊骨骼肌卫星细胞。为验证其多能性,将分离得到的卫星细胞向成肌、成脂和成骨三个方向诱导。成肌诱导后形成明显的多核肌管细胞,标记基因MyoG表达阳性,快肌肌球蛋白细胞免疫荧光染色呈阳性；成骨诱导后经茜素红和ALP染色细胞均呈阳性,成骨细胞特异性基因Osteocalcin表达阳性；成脂诱导后细胞周围有明显的脂滴出现,经0-油红染色呈阳性,且PPARy2表达呈阳性。说明本实验得到的细胞具有一定的多能性。本研究初步建立了绵羊骨骼肌卫星细胞体外分离、纯化、培养和鉴定的方法。
[Abstract]:Skeletal muscle satellite cells are adult myogenic stem cells.Although satellite cells have been successfully isolated from skeletal muscles of some species, such as mice, rats and humans, little has been done on livestock (such as cattle and sheep).It is necessary to identify and verify the gene expression of transgenic cell lines before animal production.Therefore, the establishment of animal skeletal muscle satellite cells in vitro culture model is particularly urgent and important.In this study, sheep skeletal muscle satellite cells were selected as the research object. The isolation, purification and in vitro culture of the satellite cells were discussed, and the obtained cells were identified.The purpose of this study was to establish a stable passage sheep satellite cell line and its identification method, and to provide experimental evidence for the future research and application of sheep skeletal muscle satellite cells in animal breeding and regenerative medicine.Sheep skeletal muscle satellite cells were isolated and purified by two-step enzyme digestion method and differential adherence method.The results showed that the separation efficiency of sheep skeletal muscle satellite cells by two-step digestion of 1. 1% collagenase and 0.25% trypsin was higher, and the culture medium containing 20s 10% horse serum was more favorable for the in vitro culture of sheep skeletal muscle satellite cells.The expression of several marker genes Desmin, 伪 -SarcOmeric Actinin 1 (MyOD1) Myf5 and PAX7 in sheep skeletal muscle satellite cells were detected by immunohistochemistry and flow cytometry.The results showed that the isolated cells were sheep skeletal muscle satellite cells.To verify its pluripotency, the isolated satellite cells were induced in three directions: myoblast, fat-forming and osteogenesis.After induction of myogenesis, the myoblast cells were formed, the labeled gene MyoG was positive, the fast myosin cells were positive by immunofluorescence staining, and the cells were positive by alizarin red and ALP staining after osteogenesis induction.The expression of specific gene Osteocalcin in osteoblasts was positive, and there were obvious lipid droplets around the cells after fat-forming induction, and the expression of PPARy2 was positive after 0-oil red staining.The results show that the cells obtained in this experiment have certain pluripotency.In this study, a method for isolation, purification, culture and identification of sheep skeletal muscle satellite cells in vitro was established.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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