肉羊骨骼肌卫星细胞的培养及MRFs基因表达量的分析

发布时间：2018-03-12 13:43

本文选题：骨骼肌卫星细胞　切入点：MRFs　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：骨骼肌卫星细胞是一类具有增殖能力和分化潜力的生肌干细胞,它存在于肌细胞膜和基膜两者之间,这类细胞对于以后骨骼肌的成长和再生起着重要的作用。该论文从内蒙古锡林郭勒盟采集乌珠穆沁羊的胎儿期(3月龄)及刚出生(0月龄)、中年羊(6月龄)肌肉样品,采用两步酶消化法和组织块法对骨骼肌卫星细胞进行体外提取培养;用1%的明胶和4%的多聚甲醛两种助贴壁剂来检测细胞的贴壁时间;利用差速贴壁法、peroll密度梯度离心法和改良后的纯化方法来进行细胞纯化;并用免疫组化鉴定了骨骼肌卫星细胞;用实时荧光定量技术检测了骨骼肌细胞在不同生长时期MRFs基因表达变化。得出如下结论:以组织块法分离和改良后的纯化方法纯化并用1%的明胶作为助贴壁剂得到的细胞数目较多且形态较好、细胞较纯,建立了一套高效稳定的骨骼肌卫星细胞分离纯化体系。用desmin和myosine抗体对该细胞进行鉴定,细胞质中的胞浆被染成棕色或者棕黄色,证实此细胞为骨骼肌卫星细胞。MRFs家族基因中MyoD和Myf5主要在骨骼肌卫星细胞的增殖期表达,MyoG和Myf6主要在细胞的分化阶段表达。其中MyoD和Myf5在细胞增殖期表达量上升,而到分化期时表达量下降;MyoG和Myf6正好相反,在细胞增殖期表达量下降,而到分化期时表达量上升。
[Abstract]:Skeletal muscle satellite cells are a class of myogenic stem cells with proliferative and differentiation potential, which exist between the muscle cell membrane and the basement membrane. These cells play an important role in the growth and regeneration of skeletal muscle in the future. Two step enzyme digestion method and tissue mass method were used to extract and culture skeletal muscle satellite cells in vitro, and two kinds of adherent agents (1%% gelatin and 4%% paraformaldehyde) were used to detect the adherence time of the cells. The cell was purified by differential adherent density gradient centrifugation and modified purification method, and the skeletal muscle satellite cells were identified by immunohistochemistry. The changes of MRFs gene expression in skeletal muscle cells at different growth stages were detected by real-time fluorescence quantitative technique. The conclusions are as follows: the method of tissue mass separation and purification was used to purify skeletal muscle cells and gelatin 1% was used as adherent agent. To a larger number of cells and a better shape, A set of high efficient and stable isolation and purification system of skeletal muscle satellite cells was established. The cells were identified by desmin and myosine antibodies. The cytoplasm of the cells was stained brown or brown. It was confirmed that the expression of MyoD and Myf5 in skeletal muscle satellite cells. MRFs family genes were mainly expressed in the proliferative phase of skeletal muscle satellite cells. MyoG and Myf6 were mainly expressed in the differentiation phase of skeletal muscle satellite cells, and the expression of MyoD and Myf5 increased in the proliferative phase of the cells. On the contrary, the expression of MyoG and Myf6 decreased at the stage of cell proliferation, but increased at the stage of differentiation.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS251.1

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