Abstract：Meat quality is affected by the type and percent of muscle fiber which is the basic unit of muscle tissue. In order to enrich the in vitro research mode of skeletal muscle growth and development, this study selected the hind-limb extensor digitorum longus of a newborn Landrace piglet (Sus scrofa) as sample, and the single muscle fiber was isolated through collagenase digestion. The type of myofiber was then accurately detected by RT-PCR and immunofluoresence. The single muscle fiber were isolated by collagenase digestion and these muscle fibers had smooth surface and the cells adhered to the myofibers originally, which indicated RNA was qualified for subsequent analysis. The result of RT-PCR indicated that myosin heavy chain Ⅱb (MyHC Ⅱb) mainly expressed in 8 myofibers. Furthermore, the result was confirmed by immunofluorescence with the antibody of myosin heavy chain I (MyHCⅠ). In this study, one simple and cheap detection method for isolation and identification of single muscle fibers of porcine skeletal muscle was established, and the result provides a muscle fiber model, which will be useful for studying the various functions and cellular processes of mature muscles in vitro.
1. 陈佩林，朱道立.2003. 骨骼肌琥珀酸脱氢酶的组织化学方法探讨[J]. 中国兽医杂志, 39（2）：10-11 (Chen P L, Zhu D L. 2003. Histochemical methods of succinate dehydrogenase in skeletal muscle [J]. Chinese Journal of Veterinary Medicine, 39 (2): 10-11)2. 李伯江，李平华，吴望军，等. 2014. 骨骼肌肌纤维形成机制的研究进展[J],中国农业科学,47(6):1200-1207.( Li B J, Li P H, Wu W J, et al. 2014. Skeletal muscle muscle fiber formation mechanism of the research progress [J]. Chinese Agricultural Sciences, 47 (6): 1200-1207.)3. 刘志军, 董佩佩, 贾俊静, 等. 2009. 肌球蛋白重链基因与肉品质的关系[J]. 畜牧与饲料科学, 30(3): 24-27.( Liu Z J, Dong P P, Jia J J, et al. 2009. The relationship between myosin heavy chain gene and meat quality [J]. Journal of Animal Science and Veterinary Medicine, 30(3): 24-27)4. 刘月光，史新娥，沈清武，等. 2011. 利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化[J]. 农业生物技术学报, 19(5):856-863.( Liu Y G, Shi X E, Shen Q W, et al. 2011. Isolation and culture of porcine skeletal muscle satellite cells and their myogenic differentiation[J]. Journal of Agricultural Biotechnology, 19(5):856-863.)5. 杨晓静.2004. 猪骨骼肌生长及肌纤维类型分布的分子机理研究[D]. 博士学位论文, 南京农业大学, 导师：赵茹茜，陈杰 .( Yang X J.2004.The molecular mechanism of pig skeletal muscle growth and muscle fiber type distribution [D]. Thesis for Ph D, Nanjing Agricultural University, Supervisor: Zhao R, Chen J.)6. 张丽, 孙宝忠. 2013. 肌纤维类型与生理代谢, 生长性能及肉质的关系[J]. 肉类研究, 27(8): 25-30.( Liao L, Sun B Z. 2013. The relationship between muscle fiber type and physiological metabolism, growth performance and meat quality [J]. Meat Research, 27 (8): 25-30.)7. 张萌，文剑明. 2007. ATP酶组织化学滴染法染色技术[J]. 解剖学研究, 29(5):396-397.( Zhang M, Wen J J. 2007. ATPase Histochemical Dyeing Dyeing Technique [J]. Anatomical Study, 29 (5): 396-397)8. 朱砺, 李学伟, 李芳琼, 等. 2002. 肌纤维生长发育规律的研究[J]. 四川农业大学学报, 20(1): 46-48.( Zhu L, Li X W, Li F Q, et al. 2002. Study on the Growth and Development of Muscle Fiber [J]. Journal of Sichuan Agricultural University, 20 (1): 46-48)9. Augusto V, Padovani C R, Eduardo G, et al. 2004. Skeletal muscle fiber types in C57BL6J mice[J]，Braz. J. morphol. Sci. 21(2): 89-94.10. Brooke M H, Kaiser K K. 1970. Three "myosin adenosine triphosphatase" systems: the nature of their pH lability and sulfhydryl dependence [J]. Journal of Histochemistry & Cytochemistry Official Journal of the Histochemistry Society, 18(18):670-672.11. Gehlert S, Weber S, Weidmann B, et al. 2012. Cycling exercise-induced myofiber transitions in skeletal muscle depend on basal fiber type distribution [J]. European journal of applied physiology, 112(7): 2393-2402.12. Keire P, Shearer A, Shefer G, et al. 2013. Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells[J]. Basic Cell Culture Protocols, 946: 431-468. 13. LaFramboise WA, Daood M J, Guthrie R D, et al. 1990. Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle[J]. Biochimica et Biophysica Acta, 1035(1): 109-112.14. Pasut A, Jones A E, Rudnicki M A. 2013. Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle[J]. Journal of Visualized Experiments Jove, 22(73):e50074-e50074.15. Ryu Y C, Kim B C. 2006. Comparison of histochemical charateristics in various pork groups categorized by postmortem metabolic rate and pork quality[J]. Journal of Animal Science, 84(4):894-901.16. Schiaffino S, Gorza L, Sartore S, et al. 1989. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres[J]. Journal of Muscle Research and Cell Motility, 10(3):197-205.17. Smerdu V, Karsch-Mizrachi I, Campione M, et al. 1994.Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle[J]. The American journal of physiology, 267(6 Pt 1):C1723-8.18. St?l P, Eriksson PO, Schiaffino S, Butler-Browne GS, et al. 1994. Differences in myosin composition between human oro-facial, masticatory and limb muscles: enzyme-, immunohisto- and biochemical studies[J]. Journal of Muscle Research and Cell Motility, 15(5):517-34.19. Verma M, Asakura A. 2011. Efficient single muscle fiber isolation from alcohol-fixed adult muscle following β-galactosidase staining for satellite cell detection[J]. Journal of Histochemistry & Cytochemistry, 59(1):60-7.20. Vogler T O, Gadek K E, Cadwallader A B, et al. 2016. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells[J]. Methods in Molecular Biology, 1460:141-62.
