【作者】 李玥；

【导师】 赵茹茜；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2008， 博士

【摘要】 本研究以肉鸡为模型,研究不同生长阶段限饲（1～14 d和50～63 d隔日限饲）对腓肠肌肌纤维肥大和类型转化的即时和长期影响,以探讨早期限饲可能的代谢程序化作甩;通过检测血清激素水平的变化,甲状腺轴和生长轴相关基因,肌肉蛋白代谢与肌纤维类型相关基因,以及凋亡相关基因在腓肠肌的表达,进一步分析限饲影响肌肉生长的可能机制;在此基础上通过分离卫星细胞并进行体外培养,深入探讨早期限饲对卫星细胞相对活力以及卫星细胞相关功能基因表达的影响,通过在培养体系中添加T₃,观察早期限饲以及限饲后恢复采食状态下卫星细胞对T₃应答反应的差异,指出早期限饲可程序化地影响肌纤维肥大,并且这种影响主要由甲状腺轴和生长轴相关基因介导,而主要作用靶细胞是卫星细胞。1限饲对肉鸡腓肠肌生长的影响本实验选取1 d三黄雏鸡,随机分为自由采食对照组（Con）、1～14 d隔日饲喂的早期限饲组（EFR）和50～63 d隔日饲喂的后期限饲组（LFR）,分别于14、63 d采集腓肠肌外侧头和血液样品,检测不同阶段限饲对内鸡整体和肌肉生长的影响。结果表明:1)早期限饲组体重及腓肠肌外侧头重持续低于对照组（除9 w体重P＜0.05、差异显著外,其余均为P＜0.01、差异极显著）,后期限饲组63 d腓肠肌重显著低于对照组（P＜0.05）,但体重与对照组没有差异。2)早期限饲组14d血清T₃、T₄水平均显著低于对照组（P＜0.01）,63d血清T₃水平仍显著低于对照组（P＜0.01）;后期限饲组63 d T₃、T₄与对照组无差异。3)早期限饲主要降低了直径较粗的腓肠肌快肌纤维的横截面积,且具有长期作用。后期限饲对所有肌纤维平均横截面积没有影响。4)14 d早期限饲组慢肌及快红肌肌球蛋白重链mRN脓达水平显著高于对照组（分别为P＜0.01和P＜0.05）,快白肌肌球蛋白重链基因表达显著低于对照组（P＜0.05）;63 d早期限饲组慢肌肌球蛋白重链mRNA基因表达显著低于对照组（P＜0.05）,快红肌和快白肌肌球蛋白重链与对照组相比均无差异（P＞0.05）。5)ATPase染色法检测到早期限饲组14d慢肌纤维含量增加,63 d慢肌纤维含量降低,快肌纤维含量增加。6)两种方法检测后期限饲组肌纤维类型均无变化。结果表明:1)早期限饲和后期限饲对肉鸡腓肠肌生长影响截然不同,早期限饲的影响较为显著并且持久,表现“程序化”作用;2)早期限饲阻碍了肉仔鸡肌纤维类型的转化,抑制了肌纤维的肥大,延缓了肌肉的发育及增重,最终降低肉鸡体重。3)甲状腺激素可能参与早期限饲程序化作用的调节。2限饲影响肉鸡腓肠肌生长的分子机理研究为了进一步探索早期限饲对肌纤维生长程序化作用的可能机理,本实验于14、63d采集垂体、肝脏及腓肠肌外侧头样品（用于分子生物学鉴定）及腓肠肌组织样,检测不同阶段限饲对生长轴和甲状腺轴相关基因、涉及蛋白代谢和肌纤维类型转化的相关候选基因（Calpain3,PGC-1,Calcineurin,NFAT）表达的影响,并对14 d肌肉腓肠肌组织的细胞增殖和凋亡情况进行了对比。结果表明:1)与对照组相比,早期限饲组14d肝脏GHR mRNA表达下调（P＜0.05）,腓肠肌IGFR-ImRNA表达上调（P＜0.05）,IGF-ImRNA表达下调（P＜0.05）,63d肝脏TRα表达上调（P＜0.05）,腓肠肌GHR和IGFR-I mRNA表达上调（P＜0.05）;后期限饲组除63d肝脏GHR mRNA表达上调（P＜0.05）外,其余基本没有变化;腓肠肌TRα表达在各组间均无差异。2)早期限饲组PCNA蛋白阳性较强的肌纤维极显著低于对照组（P＜0.01）,腓肠肌中BaxmRNA表达及Bax mRNA/Bcl-2 mRNA均显著高于对照组（P＜0.05）,暗示早期限饲组凋亡增加。3)早期限饲、后期限饲不影响4种蛋白代谢和肌纤维类型转化相关基因的表达。结果表明:1)早期和后期限饲通过截然不同的机制影响内分泌系统,早期限饲对肉鸡的代谢程序化影响涉及到各级组织中内分泌轴相关基因表达的改变。2)早期限饲抑制了14日龄肉鸡腓肠肌卫星细胞的增殖且可能促进了整个肌肉组织中细胞的凋亡。2)限饲不影响腓肠肌中相关蛋白代谢和肌纤维类型转化候选基因的表达,提示肌纤维类型的变化可能是肌纤维肥大受阻的继发作用。3早期限饲及恢复采食对腓肠肌卫星细胞增殖能力及内分泌轴基因表达的影响鉴于卫星细胞增殖是肌纤维肥大的必要条件,本试验选取1 d三黄雏鸡,随机分为三组（对照组、14天隔日限饲组与12天隔日限饲2天恢复组）,于14 d提取腓肠肌外侧头卫星细胞,荧光半定量PCR法检测其生长轴、甲状腺轴及凋亡相关基因的表达,并通过体外培养添加不同浓度（3ng/mL、6ng/mL和生理浓度12ng/mL）的T₃观测各组卫星细胞的增殖状况。结果表明:1)14天限饲组卫星细胞IGF-ImRNA表达显著低于12天限饲2天恢复组（P＜0.05）,极显著低于对照组（P＜0.01）,12天限饲2天恢复组表达低于对照组（P=0.06）;GHR mRNA表达对照组极显著高于14天限饲组（P＜0.01）、显著高于12天限饲2天恢复组（P＜0.05）,后两组间没有差异（P＞0.05）;IGFR-ImRNA表达在14天限饲组、12天限饲2天恢复组均显著高于对照组（P＜0.05）,但后两组间没有差异（P＞0.05）。2)卫星细胞TRαmRNA表达在14天限饲组最高,与对照组差异显著（P＜0.05）、与12天限饲2天恢复组差异极显著（P＜0.01）,对照组与12天限饲2天恢复组间差异不显著。3)Bax、Bcl-2 mRNA表达在3组间没有差异,但Bax mRNA/Bcl-2mRNA 14天限饲组最高、12天限饲2天恢复组最低,两组间差异显著（P＜0.05）,但二组与对照组间均没有差异。4)体外培养MTT法测定卫星细胞活力和形态学观察结果一致,3组卫星细胞活力对照组最强,12天限饲2天恢复组其次,14天限饲组增殖能力非常弱。5)各浓度的T₃处理对于14天限饲组卫星细胞均无影响;对照组卫星细胞在12 ng/mL T₃处理下细胞活力24、48h时极显著高于空白对照组（P＜0.01）,72 h时与对照组无差异;12天限饲2天恢复组卫星细胞在6 ng/mL T₃处理组48、72 h时细胞活力均显著高于空白对照组（P＜0.01）。结果表明:1)早期限饲显著降低了卫星细胞的相对活力,限饲后恢复自由采食可抑制凋亡并部分恢复卫星细胞的活力。2)内分泌轴相关基因的表达与卫星细胞活力相吻合,提示参与卫星细胞功能的调节。3)T₃处理3天后,低于正常生理水平的T₃（6 ng/mL）对限饲恢复后的卫星细胞的活力有促进作用,而正常生理水平的T₃（12 ng/mL）对于对照组（正常）的卫星细胞的活力已经没有促进作用,提示早期限饲改变卫星细胞对T₃反应的时间和剂量依赖性,可能参与补偿性生长的调控。更多还原

【Abstract】 The present study comprises three series of experiments designed to describe the effect of feed restriction during different developmental stages on morphology and function of myofibers and satellite cells,and to elucidate the possible underlying mechanisms involved in such effect,using broiler chickens as model.（1） the body and muscle growth retardation and alterations in myofiber hypertrophy and myofiber type transformation induced by early（1～14 d） and late（50～63 d） feed restriction were compared to reveal the role of nutritional deficiency during early posthatch development in programming the morphology and function myofibres in gastrocnemius;（2） the serum levels of hormones,as well as the hepatic and muscle expression of genes involved in GH/IGF-1 and thyroid hormones targeting,protein metabolism and myofiber type transformation,as well as cell apoptosis,were determined to explore the possible mechanism underlying the effect of feed restriction on muscle growth;（3） satellite cells were isolated from muscle of control,restricted, and refed chickens and cultured in vitro to elucidate the effect of in vivo dietary intervention on proliferation and gene expression of satellite cells and their responses to different levels of thyroid hormone in vitro.1 Effect of feed restriction on morphology of gastrocnemius muscle in broiler chickens1-day-old（1 d） broiler chickens were allocated randomly into control（Con）, early feed restriction（EFR） and late feed restriction（LFR） groups.Chickens of Con group were fed ad libitum throughout the experiment for 9 weeks,while chickens of EFR and LFR groups were subjected to intermittent feed restriction with feed provided on alternate days for the first and the last 2 weeks of their posthatch life, respectively.Chickens from each group were sacrificed at 14 d and 63 d.Serum thyroid hormone concentration were measured and the lateral gastroenemius muscle were taken for measuring the cross-sectional areas and densities of each type of myofibers using H.E staining,myosin ATPase staining and myofiber typing by relative quantification of mRNA expression for three different types of myosin heavy chain（MyHC）:slow myofiber MyHC（SM MyHC）,red fast-twitch myofiber MyHC （FRM MyHC） and white fast-twitch myofiber MyHC（FWM MyHC）,in order to investigate the influence of feed restriction during different developmental stages on muscle growth.The results showed that 1) Chickens of EFR group exhibited consistently lower body weight throughout the experiment,while chickens of LFR group only showed a transient decrease in body weight on 56 d but caught up with their counterparts in Con group by 63 d.However,the lateral gastrocnemius muscle weights in both EFR and LFR groups were significantly lower compared with those in Con group of the same age.2) The serum T₃,T₄ levels were significandy lower in EFR on 14 d while only T₃ remained low by 63 d.LFR did not affect serum T₃ or T₄ levels.3) LFR group had no effect on the cross-sectional areas of the total myofibers while the cross-sectional areas of fast-twitch myofibres were both decreased in EFR group on both 14 d and 63 d.4) EFR group exhibited significantly higher SM and FRM MyHC mRNA expression but lower FWM MyHC mRNA compared with their control counterparts on 14 d.On 63 d however,a significant down-regulation was observed in EFR group for SM MyHC expression,whereas no significant differences were detected for both FRM and FWM MyHC mRNA expression between EFR and Con groups.5) Histological study by ATPase staining revealed that the percentage of slow myofibers increased on 14 d while decreased on 63 d in EFR group,and that of the fast myofibers increased significantly on 63 d.6) No alteration in percentages of slow and fast fibers was observed for LFR group compared with the control in both measurements.These results indicate that 1) the influences of early and late feed restriction on gastrocnemius muscle growth in broilers are different;2) early feed restriction may delay the conversion from slow-tonic myofiber to fast-twitch myofiber and inhibit myofibers’ hypertrophy,resulting in retarded muscle growth that may account for the decreased body weight,demonstrating more profound and lasting effects on broiler growth;3) thyroid hormones might participate in mediating such programming effect.2 Mechanisms involved in the effect of feed restriction on gastrocnemius muscle growth in broiler chickensIn order to explore the mechanisms underlying the effect of feed restriction on gastrocnemius muscle growth,pituitary,liver and the lateral gastrocnemius muscle were taken and abundances of mRNA for genes involved in GH/IGF-1 and thyroid hormones targeting（growth hormone（GH）,growth hormone receptor（GHR）, insulin-like growth factor-Ⅰ（IGF-Ⅰ）,type-ⅠIGF receptor（IGFR-Ⅰ）,thyroid hormone receptorα（TRα） and thyroid hormone receptorβ（TRβ））,protein metabolism and myofiber typing（peroxisome proliferator-activated receptor-γcoactivator-1（PGC-1）, calcineurin,nuclear factor of activated T cells（NFAT） and calpain3）),cell proliferation or apoptosis（Pax7,B cell lympfoma/leukemia 2（Bcl-2）,Bcl-2 associated X protein（Bax）） were determined by RT-PCR with 18S rRNA as an internal-standard. In addition,genomic DNA was isolated from 14 d lateral gastrocnemius and subjected to electrophoresis for the evaluation of apoptosis（DNA Ladder）,and fluorescent immunostaining of PCNA were carded out for morphological identification of proliferating satellite cells.The results showed that 1) chickens of EFR group exhibited significantly lower GHR mRNA expression in liver,significantly lower IGF-Ⅰbut higher IGFR-ⅠmRNA expression in gastrocnemius muscle at 14 d,and significantly higher TRαin liver, significantly higher GHR and IGFR-ⅠmRNA expression in gastrocnemius muscle at 63 d.2) Bax mRNA expression and Bax mRNA/Bcl-2 mRNA were significantly higher in EFR group at 14 d;and 3) the density of PCNA positive cells was significanfly lower in EFR group.4) LFR group did not show significant alterations in mRNA of any genes investigated except higher GHR mRNA expression in liver at 63 d.5) No changes in PGC-1,Calcineurin,NFAT,Calpain3 mRNA expression were detected in group ERF and LRF on either ages.These results indicate that 1) early feed restriction exerts metabolic programming effect involving changes of mRNA expression for the target genes of GH/IGF-1 and thyroid hormone,while late feed restriction has only transient and minor influence. Distinct mechanisms seem to apply for the effects of feed restriction during different periods of life.2) Early feed restriction inhibited proliferation of satellite cells and stimulated apoptosis in gastrocnemius muscle of broiler chickens at 14 d,indicating that satellite cells may be the target for this programming effect of early nutritional deficiency on gastrocnemius muscle growth.3 Effect of early feed restriction and refeeding on satellite cells activity and endocrine-related genes expression on satellite cells in gastrocnemius muscle of Broiler Chickens To elucidate whether the programming effect of early feed restriction on myofiber development is achieved via targeting on satellite cells,1 d broiler chickens were allocated randomly into control（14 days feed ad libitum）（Con）,14 days feed restriction（14FR） and 12 days feed restriction folllowed by 2 days refeeding （12FR2R） groups.14FR and 12FR2R groups were subjected to intermittent feed restriction with feed provided on alternate days for the first 14 days and 12 days, respectively.Satellite cells were isolated from the lateral gastrocnemius of each group at 14 d,RNA of isolated satellite cells were extracted and abundances of GHR,IGF-Ⅰ, IGFR-Ⅰ,TRα,Bcl-2 and Bax mRNA were determined by real-time quantitative PCR usingβ-actin as an internal-standard.Morphological changes of isolated satellite cells from each group were observed for 3 days in culture and MTT assay was performed to assess the cell viability.T₃ was added to the culture of satellite cells from each group at the level of 12 ng/mL（physiological level）,3 ng/mL and 6ng/mL to investigate the changes of satellite cells viability exposed to different concentrations of T₃.The results showed that 1) IGF-Ⅰand GHR mRNA expression were decreased in 14FR group,while IGFR-ⅠmRNA expression increased.2) The expression of TRαmRNA was significantly increased in 14FR group compared to Con and 12FR2R groups,and there was no significant difference between other two groups.3) Bax mRNA/Bcl-2 mRNA was significantly lower in 12FR2R group than in 14FR group. 4) Satellite cells of Con group exhibited highest viability,while that of 14FR group showed the lowest viability with that of 12FR2R group in between.5) Satellite cells of 14FR group showed no response to any concentrations of T₃,while cells in Con group increased viability when treated with 12 ng/mL T₃ for 48 h but not 72 h. 12FR2R group seems to respond to lower T₃ at 6 ng/mL for 48 h and 72 h.These results indicate that 1) early feed restriction may inhibit the proliferation and stimulate apoptosis of satellite ceils,and refeeding may partly restore it.2) Genes involved in GH/IGF-1 and thyroid hormone targeting may mediate this effect,and 3) early feed restriction may increase the sensitivity or reception of satellite cells to T₃ after refeeding,contributing to compensatory muscle growth.更多还原