【作者】 杨秋梅；

【导师】 杨公社；

【作者基本信息】 西北农林科技大学 ， 动物学， 2011， 硕士

【摘要】 骨骼肌纤维类型及数量影响家畜肉品质。根据其代谢和收缩功能,猪生后骨骼肌纤维分为慢速氧化型、快速氧化型、快速酵解型和中间型四种,分别由不同的肌球蛋白重链(myosin heavy chain,MyHC)异构体MyHC-I、MyHC-II2a、MyHC-II2b和MyHC-II2x确定。猪骨骼肌纤维数目在出生前已确定,在生后的生长发育过程中,主要是骨骼肌卫星细胞的分化及肌纤维类型的转变。研究如何有效控制猪骨骼肌卫星细胞定向分化,调控肌纤维类型组成,改善肉品质具有重要的实践意义。Wnt/β-catenin信号通路在肌肉生长、发育过程中起着重要调节作用。目前,Wnt/β-catenin信号通路调控出生后猪骨骼肌纤维类型组成作用还未见报道。因此,本研究利用冰冻切片HE染色、油红O染色、mATPase及免疫组化等组织学方法及Real time PCR、Western Blotting分子生物学技术,在明确猪骨骼肌发育的组织形态学特征和组织学规律的基础上,研究Wnt/β-catenin信号通路相关基因在猪骨骼肌发育过程中的表达规律;再利用15 mMol/L LiCl处理单根肌纤维分离培养的骨骼肌卫星细胞,采用Western Blotting、免疫荧光等细胞生物学及分子生物学技术,探讨其在猪骨骼肌卫星细胞分化过程中的调控作用。主要研究结果如下:1.随着猪日龄增长,骨骼肌纤维横切面积逐渐增大,慢肌向快肌转化,慢肌纤维横切面积明显小于快肌纤维横切面积;比目鱼肌相对于背最长肌和趾长伸肌,其聚脂能力强,慢肌含量高。2.猪背最长肌发育过程MyHC-I、GSK-3β、β-catenin和Fz3 mRNA随着个体日龄增长表达量下降;MyHC-IIb mRNA表达逐渐升高;MyHC-IIa mRNA表达在1日龄~2周龄阶段升高后又降低;MyHC-IIx mRNA表达变化不显著。慢肌、β-catenin和p-GSK-3β蛋白与mRNA表达一致,而p-β-catenin、GSK-3β蛋白及快肌蛋白表达上调。β-catenin和慢肌蛋白表达量在6月龄猪比目鱼肌中表达最高,趾长伸肌中表达最低;快肌蛋白的表达量则相反;3. 15 mMol/L LiCl诱导骨骼肌卫星细胞向慢肌分化,p-GSK-3β、β-catenin、myosin及慢肌蛋白增加,p-β-catenin、GSK-3β及快肌蛋白减少,并且核内源性β-catenin增多。综上所述,随着年龄增长,骨骼肌纤维横切面积逐渐增大,且快肌纤维显著大于慢肌;比目鱼肌相对于背最长肌和趾长伸肌,其聚脂能力强,慢肌含量高。发现Wnt/β-catenin信号通路促进猪骨骼肌慢肌纤维生长。利用LiCl激活经典Wnt信号通路,在猪骨骼肌卫星细胞分化中发挥正调节作用,促进成肌分化;并诱导猪骨骼肌卫星细胞向慢肌分化。更多还原

【Abstract】 The number of skeletal muscle fiber and types have influence on the meat quality of livestock. There were divided into slow oxidative, fast oxidative, fast glycolytic and intermediate type according to the metabolism and contractile function of postnatal porcine, respectively, by different myosin heavy chain(MyHC) isormers of MyHC-I, MyHC-IIa, MyHC-IIb and MyHC-IIx determined. The number of skeletal muscle fiber were determined porcine prenatal, it be will depend on skeletal muscle satellite cells differentiation and muscle fiber type transformation in the future development. Study how to effectively control the satellite cells directional differentiation and regulate of fiber type composition to improve the meat quality has important practical significance. Wnt/β-catenin signaling pathway which plays an important role to regulate muscle growth and development. Currently, it has not been reported that the role of Wnt/β-catenin signaling pathway regulated composition of postnatal porcine skeletal muscle fiber type. Therefore, this study used histological methods, such as frozen sections of HE staining, Oil red O staining, mATPase staining, immunohistochemical staining, and molecular biology techniques of Real time PCR and Western Blotting to identify morphology and histology development regularity of porcine skeletal muscle. Studying of relate genes expression pattern in Wnt/β-catenin signaling pathway during porcine skeletal muscle development. Using 15 mMol/L LiCl treatment of satellite cells with single isolated muscle fiber. To utilize cell biology and molecular biology techniques ,such as Western Blotting and immunofluorescence staining, to investigate of Wnt/β-catenin signaling pathway in porcine satellite cells differentiation in vitro. The main results are as follows:1. The cross sectional area of skeletal muscle fiber increased during porcine development. The slow muscle transformed into fast muscle and slow muscle fiber significantly smaller than fast muscle fiber. The capacity of polyester in SOL was better than LD and EDL, and have more slow muscle fiber.2. The mRNA expression levels of MyHC-I, GSK-3β,β-catenin and Fz3 decreased with the development of LD, MyHC-IIb increased, MyHC-IIa increased from 1-day old to 2weeks then decreased, MyHC-IIx did not change significantly. The protein expression levels of slow muscle,β-catenin and p-GSK-3βas same as the mRNA, while p-β-catenin, GSK-3βand fast muscle increasd. The protein expression levels ofβ-catenin and slow muscle in SOL were the highest, there were the lowest in EDL at 6-month old, in contrast of fast muscle protein.3. 15 mMol/L LiCl induced skeletal muscle satellite cells differentiation into slow muscle fiber. The protein expression levels of p-GSK-3β,β-catenin, myosin and slow muscle increased, p-β-catenin, GSK-3βand fast muscle decreased, and the endogenousβ-catenin in nuclear increased.In summary, the skeletal muscle fiber cross sectional area gradually increased during development, and the area of fast muscle fiber was significantly larger than slow. The capacity of polyester in SOL was better than LD and EDL, and have more slow muscle fiber. The result found that Wnt/β-catenin signaling pathway promotes the porcine slow muscle fiber to growth. Wnt signaling pathway promoted satellite cells to myogenic differentiation when it was activitied by LiCl, and induced satellite cells differentiation to slow muscle fiber.更多还原