1、 猪论文：Wnt/-catenin 信号通路调控猪骨骼肌纤维类型变化的初步研究【中文摘要】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/-catenin
2、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/-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/-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
3、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-3、-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、-catenin 和 p-GSK-3 蛋白与 mRNA 表达一致,而 p-catenin、G
4、SK-3 蛋白及快肌蛋白表达上调。-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-3、-catenin、myosin 及慢肌蛋白增加,p-catenin、GSK-3 及快肌蛋白减少,并且核内源性 -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/-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
5、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。【英文摘要】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 p
6、ostnatal 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 trans
7、formation 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/-catenin signaling pathway which plays an important role to regulate musc
8、le growth and development. Currently, it has not been reported that the role of Wnt/-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 s
9、taining, 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/-catenin signaling pathway during porcine skeletal
10、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/-catenin signaling pathway in porcine satellite cells dif
11、ferentiation 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 bett
12、er than LD and EDL, and have more slow muscle fiber.2. The mRNA expression levels of MyHC-I, GSK-3,-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 leve
13、ls of slow muscle,-catenin and p-GSK-3as same as the mRNA, while p-catenin, GSK-3and fast muscle increasd. The protein expression levels of-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 skele
14、tal muscle satellite cells differentiation into slow muscle fiber. The protein expression levels of p-GSK-3,-catenin, myosin and slow muscle increased, p-catenin, GSK-3and fast muscle decreased, and the endogenous-catenin in nuclear increased.In summary, the skeletal muscle fiber cross sectional are
15、a 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/-catenin signaling pathway promotes the porcine slow muscle fiber to
16、 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.【关键词】猪 Wnt/-catenin 信号通路 肌纤维类型 卫星细胞 分化【英文关键词】Porcine Wnt/-catenin signaling pathway fiber types satellite cell differen
17、tiation【目录】Wnt/-catenin 信号通路调控猪骨骼肌纤维类型变化的初步研究 摘要 6-8 ABSTRACT 8-9 文献综述 12-27 第一章 骨骼肌生长发育及再生概述 12-18 1.1 骨骼肌生长发育过程 12-15 1.1.1体节发育 12-13 1.1.2 生肌节发育 13 1.1.3 肌纤维形成 13-14 1.1.4 骨骼肌纤维类型的分类 14-15 1.2 骨骼肌再生 15-18 1.2.1 肌卫星细胞的起源及特征 15-16 1.2.2. 调控卫星细胞分化的转录因子 16-18 第二章 Wnt 信号与骨骼肌发育 18-27 2.1 Wnt 及信号通路 18-2
18、3 2.1.1 Wnt 家族 18-19 2.1.2 多条 Wnt 信号途径 19-23 2.2 Wnt/-catenin 信号通路在骨骼肌发育过程中的作用 23-25 2.3 Wnt/-catenin 在骨骼肌纤维类型生长过程中的作用 25-27 试验研究 27-60 前言 27-28 第三章 猪骨骼肌发育过程中的组织形态学研究 28-34 3.1 实验材料与方法 28 3.1.1 实验动物 28 3.1.2 主要试剂及配方 28 3.1.3 实验仪器与器械 28 3.1.4 实验材料及冰冻切片制备 28 3.1.5 主要的实验方法 28 3.2结果与分析 28-32 3.2.1 冰冻切片
19、HE 染色 28-30 3.2.2 冰冻切片油红 O 染色 30-31 3.2.3冰冻切片免疫组化染色 31-32 3.2.4 冰冻切片 m ATPase 染色 32 3.3 讨论 32-33 3.4 小结 33-34 第四章 猪骨骼肌发育过程中 Wnt 34-45 4.1 材料与方法 34-35 4.1.1 实验动物 34 4.1.2 主要试剂及配方 34 4.1.3 主要实验仪器及设备 34 4.1.4 主要实验方法 34-35 4.1.5 统计分析 35 4.2 结果 35-42 4.2.1 猪背最长肌发育过程中 Wnt/-catenin 信号通路相关基因与 MyHCs 的表达规律 35
20、-41 4.2.2 猪不同部位骨骼肌中 -catenin 与 MyHCs 的表达规律 41-42 4.3 讨论 42-44 4.4 小结 44-45 第五章 Wnt/-catenin 信号通路对猪骨骼肌卫星细胞分化为快、慢肌的影响 45-60 5.1 材料与方法 45-46 5.1.1 实验动物 45 5.1.2 主要试剂及配方 45 5.1.3 主要仪器设备 45 5.1.4 主要的实验方法 45-46 5.1.5 统计分析 46 5.2 结果与分析 46-57 5.2.1 猪骨骼肌卫星细胞的鉴定 46-47 5.2.2 LiCl 处理猪骨骼肌卫星细胞最佳浓度筛选 47-48 5.2.3 LiCl 处理后 Wnt/-catenin 信号通路相关因子的蛋白表达 48-50 5.2.4 激活 Wnt/-catenin 信号通路促进猪骨骼肌卫星细胞分化 50-54 5.2.5 LiCl 处理后促进猪骨骼肌卫星细胞向慢肌分化 54-57 5.3 讨论 57-59 5.4 小结 59-60 结论 60 创新点 60 有待进一步完善及深入研究的问题 60-61 参考文献 61-71 附录 71-79 缩略词表 79-80 致谢 80-81 作者简介 81
