IGF-I-CaN-NFATc3信号通路相关基因在鸭发育早期骨骼肌中的同步表达及其与肌纤维性状相关性

doi:10.3864/j.issn.0578-1752.2015.06.15

摘要/Abstract

摘要： 【目的】IGF-I-钙调磷酸酶（CaN）-NFAT信号途径是调节肌肉生长、决定不同肌纤维类型的主要信号途径，本研究选择生长速度不同的高邮鸭和金定鸭为试验材料，首次对不同品种鸭胚胎期和出雏早期肌肉中IGF-I-CaN-NFAT信号通路相关基因的表达规律及其与肌纤维类型的相关性进行了研究。【方法】采用实时荧光定量PCR方法研究鸭13、17、21、25、27胚龄和出雏后7日龄胰岛素样生长因子( insulin-like growth factors, IGF-I) , 钙调磷酸酶（calcineurin, CnAα）和活化T细胞核因子（nuclear factor of activated T cells, NFAT c3）基因的表达量，同时采用肌球蛋白ATPase碱孵育法对两个鸭种腓肠肌外侧头不同发育阶段的肌纤维性状进行组织学观察，对不同类型肌纤维比例进行统计，用SPSS20.0软件进行差异显著性分析，探讨IGF-I-CaN-NFAT信号通路相关基因对鸭骨骼肌肌纤维类型转换的调控机制。【结果】随着胚龄或日龄增加，两鸭种快肌纤维比例均呈现下降趋势，中间型肌纤维比例仅存在少量波动，慢肌纤维比例除在高邮鸭中27胚龄时有所下降外，总体上呈现上升的趋势；IGF-I, CnAα和 NFATc3 基因在不同鸭种的同一肌肉部位的表达模式相对一致，仅存在显著的发育时段差异，在不同的部位（胸肌和腿肌）的表达也存在差异，但并不存在显著的性别效应，3个基因均在13胚龄即可检测到表达，且表达量均表现为最高，显著地高于其他各胚龄的表达量，提示IGF-I-CaN-NFATc3信号通路可能在鸭胚胎发育早期肌纤维的形成过程中发挥了重要作用。CnAα mRNA在胸肌中呈近似“V”形的表达趋势，21胚龄时表达量最低然后呈持续上升趋势，而在腿肌中则呈“波浪形”表达趋势，表达量最低点出现在27胚龄；NFATc3在胸肌和腿肌中的表达量均表现为13胚龄最高，然后至17胚龄显著下降并维持在较低的表达水平，在胸肌中表达量最低点出现在21胚龄，而腿肌中21胚龄的表达量则要显著高于17、25、27胚龄和7日龄的表达量，17胚龄表达量最低；IGF-I mRNA在胸肌和腿肌中总体上表现为先下降，至出雏前（27胚龄）降至最低，然后出雏后一周略有上升的趋势。IGF-I, CnAα和 NFATc3 基因的表达两两之间均表现出显著或极显著的正相关关系，同时，3个基因的表达与快肌纤维比例存在正相关，而与慢肌纤维比例及中间型纤维的比例呈负相关。【结论】在鸭胚胎发育后期至出雏早期，腓肠肌外侧头肌纤维类型存在由快肌纤维向慢肌纤维转换的趋势，IGF-I-CaN-NFAT信号通路相关基因之间存在协同表达，在鸭早期骨骼肌纤维类型转换中可能发挥着调控作用。

关键词: 早期发育, 基因表达, 肌纤维类型, IGF-I-CaN-NFAT信号通路, 鸭

Abstract: 【Objective】Insulin-like growth factor I (IGF-I)-calcineurin (CaN)-NFAT signal pathway have been implicated in the regulation of myocyte hypertrophy and fiber type specificity. In the present study, expression of CnAα, NFATc3 and IGF-I genes was firstly studied in Gaoyou and Jinding ducks differing in their muscle growth rates. 【Method】 Expression of IGF-I, CnAα and NFATc3 was quantified by RT-PCR in the breast muscle (BM) and leg muscle (LM) on days 13, 17, 21, 25 and, 27 of embryonic development, as well as at 7 days post-hatching (PH). Lateral gastrocnemius muscles collected from two duck breeds were cut at -20℃ using a cryotome and then stained with the method of myosin-ATPase in order to analyze muscle fiber type composition. SPSS 20.0 software was used to conduct the significant difference analysis, for investigating the possible regulation mechanism of IGF-I-CaN-NFAT pathway in myofiber type transition. 【Result】 Overall, the percentage of type I fibers increased and that of type IIb fibers decreased consistently. However, the percentage of type IIa fibers was almost constant as development proceeded in both duck breeds. The consistent expression patterns of CnAα, NFATc3 and IGF-I were found in the same anatomical location at different development stages in both duck breeds, showing extremely significant differences in age-specific fashion. However, the three genes were differentially expressed in different anatomical location (BM and LM). CnAα, NFATc3 and IGF-I mRNA could be detected as early as on E 13d, and the highest level was appeared at this stage in both BM and LM, significantly higher than those at the other five development stages, indicating that these three genes might have some effects on the ontogenesis of myofibers. The mRNA expression of duck CnAα in BM showed a tendency as the “V”, the lowest level appeared on E 21d, and then increased continuously up until 7 days PH in both duck breeds, while in LM, CnAα gene expression was variable throughout the course of this study and the lowest level appeared on E 27d. Expression of NFATc3 decreased significantly to a very low level from E 13d to E 17d and maintained this relative low level during late development stages in both BM and LM, the lowest level appeared on E 21d in BM while the expression level on E 21d in LM was significantly higher (P＜0.05) than those on embryonic days 17, 25 and 27, and at 7 days PH. Expression of IGF-I declined to the lowest level just prior to hatch (E 27d), and then elevated a little on 1 week PH in both BM and LM in the two duck breeds. Significant positive relationships were observed for the expression of studied genes in BM and LM of both duck breeds. Meanwhile, the expression of these three genes were all showed positive relationships with the percentage of type IIb fibers and negative relationships with the percentage of type I fibers and type IIa fibers. 【Conclusion】The data of the study indicate that the fiber type of the lateral gastrocnemius muscle may transit from type IIb to type I during late-embryonic and PH development, and differential expression and coordinated developmental regulation of the selected genes that comprise the IGF-I-calcineurin-NFATc3 pathwayin the duck skeletal muscles during embryonic and early PH growth and development, and this signaling pathway might play a role in the regulation of the myofiber type transition.

Key words: early development, gene expression, myofiber type; IGF-I-calcineurin-NFATc3 pathway, duck

束婧婷，宋迟，徐文娟，章明，单艳菊，陈文峰，宋卫涛，陶志云，李慧芳. IGF-I-CaN-NFATc3信号通路相关基因在鸭发育早期骨骼肌中的同步表达及其与肌纤维性状相关性[J]. 中国农业科学, 2015, 48(6): 1195-1204.

SHU Jing-ting, SONG Chi, XU Wen-juan, ZHANG Ming, SHAN Yan-ju, CHEN Wen-feng, SONG Wei-tao, TAO Zhi-yun, LI Hui-fang. Expression Profile of IGF-I-calcineurin-NFATc3-Dependent Pathway Genes in Skeletal Muscles and Their Associations with Myofiber Traits During Embryonic and Early Post-Hatching Development in Ducks[J]. Scientia Agricultura Sinica, 2015, 48(6): 1195-1204.

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