【作者】 黄飞若；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本试验的目的是研究日粮中持续添加亚麻籽(富含n-3 PUFA)对生长肥育期猪肌肉生长的影响,并在细胞水平探讨了n-3 PUFA影响肌肉生长的分子机制。课题的研究内容由两部分组成,第一部分为动物试验,研究日粮中持续添加亚麻籽(富含n-3 PUFA)对生长肥育期猪肌肉块重量的影响,并在机体水平研究了基因表达与肌肉块重之间的关系;第二部分为细胞培养试验,是针对日粮中持续添加亚麻籽(富含n-3 PUFA)对生长肥育期猪肌肉生长的调控效果,在细胞水平探讨了n-3 PUFA调控肌肉生长的分子机制。第一部分,动物试验。试验采用单因子完全随机设计,选择80±4d健康去势公猪24头,按体重随机分为4组,每组6重复栏,单栏饲养。设计生长期(80-140d)及肥育期(140-170d)两种日粮,其中对照日粮由玉米、豆粕和脂肪粉等主要原料构成;试验日粮中加入10%的亚麻籽而不用脂肪粉,两种日粮等能等氮。4组猪随机接受日粮处理,其中第1组饲喂对照日粮(C组),其余3组猪分别在屠宰前30d,60d和90d饲喂试验日粮(分别为T1,T2和T3组),而其他时间饲喂对照日粮,试验期共90d。度量了各处理组胴体性状和肉质性状;以及背最长肌、后肢肌肉块重量(股四头肌、股二头肌、瓣膜肌、半腱肌、股薄肌和腰肌)的影响;采用气相色谱方法分析了饲料、肌肉以及脂肪组织中脂肪酸组成。并利用半定量RT-PCR法测定超氧化物酶体增殖激活受体(peroxisomeproliferators-activated receptor,PPARγ)及肿瘤坏死因子α(tumor necrosis factor,TNFα)在脾脏、肌肉组织和脂肪组织中的mRNA丰度,在试验结束时用ELISA试剂盒测定血清中TNFα浓度。主要研究结果如下:1.猪生长肥育期添加亚麻籽对组织中n-3PUFA富集量和肌肉块重量的影响。在生长肥育猪170日龄屠宰时,尽管各处理组背膘厚、瘦肉率、眼肌面积均没有显著影响(P＞0.05)。各处理组股二头肌重、瓣膜肌、半腱肌和腰肌重差异不显著(P＞0.05)。背最长肌、股四头肌和股薄肌重随亚麻籽添加时间的延长呈线性上升(P＜0.05)。背最长肌,皮下脂肪组织中LNA含量随亚麻籽添加时间的延长呈极显著线性上升(P＜0.01)。背最长肌和皮下脂肪组织中二十碳五烯酸(eicosapentenoic acid,EPA)和二十二碳五烯酸(Docosapentaenoic Acid,DPA)含量随添加时间的延长呈极显著线性上升(P＜0.01)。此外,肌肉组织中二十二碳六烯酸(Docosahexaenoic Acid,DHA)含量随添加时间的延长呈极显著二次曲线上升(P＜0.01)。回归分析发现背最长肌重随肌肉中n-3PUFA(R~2=0.89,P＜0.01),尤其是α-亚麻酸(α-Linolenic acid,LNA)(R~2=0.86,P＜0.01)含量的增加而呈显著的二次曲线上升。这些结果表明在生长肥育猪屠宰前30d-90d添加亚麻籽,通过提高组织中n-3PUFA富集量,促进了背最长肌、半腱肌和股薄肌的生长,提高肌肉重。2.为进一步在机体水平研究基因表达与肌肉块重之间的关系,试验分析了猪生长肥育期添加亚麻籽对PPARγ、TNFα基因表达和肌肉块重量的影响。脾脏中PPARγ的表达随亚麻籽添加时间增加呈线性增加(P＜0.01),肌肉组织中则呈二次曲线增加(P＜0.01),但脂肪组织中PPARγ的表达量没有受到亚麻籽添加的影响(P=0.095)。随着亚麻籽添加时间的延长,脾脏中的TNFαmRNA丰度呈线性降低(P＜0.01),肌肉组织中(P＜0.01)和脂肪组织(P＜0.01)中TNFα的mRNA丰度也随亚麻籽添加时间呈线性降低。随着亚麻籽添加时间的延长,血清中TNFα浓度线性降低(P＜0.01)。脾脏中PPARγ与TNFα(R~2=0.77,P＜0.001)的mRNA丰度有极显著的负相关。肌肉组织中PPARγ的表达量与TNFα(R~2=0.70,P=0.002)有极显著的负相关。脾脏中PPARγ的表达量与血清中TNFα浓度有极显著的负相关(R~2=0.59,P＜0.01)。肌肉组织中PPARγ的表达量与血清中TNFα浓度之间显著的负相关(R~2=0.52,P＜0.05)。回归分析发现背最长肌重分别与背最长肌中PPARγ表达量(R~2=0.80,P＜0.01)和TNFα表达量(R~2=0.87,P＜0.01)有显著的二次曲线关系。结果表明持续添加亚麻籽日粮可能以依赖PPARγ的机制下调TNFα基因表达,从而提高生长肥育猪肌肉块重量。第二部分,细胞培养试验。为了进一步研究持续添加亚麻籽日粮对生长肥育猪骨骼肌重的影响机制,体外培养C2C12小鼠骨骼肌细胞。试验分为4组,其中第1组为在培养的小鼠C2C12骨骼肌细胞中添加牛血清蛋白(Bovine serumalbumin,BSA)(正对照组),第2组为添加棕榈酸(Palmitic acid,PTA)(负对照组),第3组为添加α-亚麻酸(α-linolenic acid,LNA),第4组为添加二十碳五烯酸(eicosapentenoic acid,EPA)。在培养的小鼠C2C12骨骼肌细胞中分别添加600μM BSA、PTA、LNA和EPA,培养24小时后,细胞用100nM胰岛素培养10min,采用实时定量PCR方法测定各处理组PPARγ、TNFα及肌肉环状指基因1(muscle RING finger 1,MuRF1)基因的表达量。同时,培养的小鼠C2C12骨骼肌细胞中分别添加300μM、600μM的BSA、PTA、LNA和EPA,培养24小时后,细胞用100nM胰岛素培养10min,采用Western blot方法测定了各处理组骨骼肌细胞中IκBα蛋白量。试验进一步在培养的小鼠C2C12骨骼肌细胞中分别添加300μM、600μM的BSA、PTA、LNA和EPA,培养24小时后,细胞用100nM胰岛素培养10min,采用EMSA方法测定了各处理组骨骼肌细胞中NF-κB活性的影响。主要研究结果如下:n-3PUFA对C2C12骨骼肌细胞NF-κB活性和MuRF1基因的影响。在培养的骨骼肌细胞中持续添加的EPA(600μM,24小时)能够提高PPARγmRNA的丰度2.3倍数(P＜0.01)。LNA只提高了PPARγmRNA的丰度1.08倍。而600μM棕榈酸,培养24小时,抑制了PPARγ基因的mRNA丰度(P＜0.01)。分别在小鼠C2C12骨骼肌细胞中分别添加600μM LNA、EPA,培养24小时,TNFα基因的mRNA丰度分别被抑制了1.06、2.93倍。而棕榈酸通过抑制了PPARγ基因的mRNA丰度,提高了TNFα基因表达(P＜0.01)。小鼠C2C12骨骼肌细胞中分别添加300μM LNA、EPA,培养24小时,并没有影响到骨骼肌中IκBα的蛋白质表达量。在同样条件下,分别添加600μM LNA和EPA,LNA依然没有影响到骨骼肌中IκBα的蛋白质表达量,而IκBα的蛋白质表达量被EPA显著提高了86%(P＜0.01)。添加棕榈酸(600μM,24小时)能够降低细胞中39%IκBα蛋白质水平。分别添加600μM LNA和EPA,LNA没有影响到C2C12细胞核中NF-κB的蛋白质含量,而EPA降低了细胞核中NF-κB的蛋白质含量;添加600μM PTA提高了C2C12细胞核中NF-κB的蛋白质含量。在本试验中,分别添加600μM LNA和EPA,LNA并没有影响到骨骼肌细胞中MuRF1基因表达,而MuRF1基因表达量被EPA显著抑制了3.38倍(P＜0.01)。添加促进IκBα蛋白质降解的棕榈酸(600μM,24小时)极显著提高了MuRF1基因表达(P＜0.01)。结果表明,二十碳五烯酸通过PPARγ/TNFα/IκBα/NF-κB信号通路,抑制了MuRF1基因表达,从而减少了骨骼肌蛋白质降解,促进正常生产条件下动物骨骼肌生长。结论:1、在生长肥育猪屠宰前30d-90d添加亚麻籽,通过提高组织中n-3PUFA富集量,促进了背最长肌、半腱肌和股薄肌的生长,提高肌肉重。2、持续添加亚麻籽日粮可能以依赖PPARγ的机制下调TNFα基因表达,从而提高生长肥育猪肌肉块重量。3、二十碳五烯酸通过PPARγ/TNFα/IκBα/NF-κB信号通路,抑制了MuRF1基因表达,从而减少了骨骼肌蛋白质降解,促进正常生产条件下动物骨骼肌生长。更多还原

【Abstract】 The aim of the study was to investigated that the effect of duration of feeding linseed diet on the skeletal muslce growth of the growing-finishing pigs,and the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs.The contents of the research were mainly composed of two parts:the first part was the animal feeding experiments,which was to investigated that the effect of duration of feeding linseed diet on the skeletal muslce growth of the growing-finishing pigs and explore the relationship between gene expression and muslce mass;the second part was in vitro experiment,which investigated the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs.The fisrt part:the animal feeding experiments.Two isoenergetic and isonitrogenous diets were formulated,and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%.Twenty-four Landrace×Yorkshire barrows weighing 35±3.7 kg were randomly assigned to four treatments with six individuals per treatment.Pigs in treatment 1(T1) fed the control diet throughout the experimental period,while pigs in T2,T3 and T4 fed the control diet except for 30,60,and 90 d prior to slaughter when the linseed diet were fed.The experiment was conducted for 90 days.Carcass quality and meat quality data were collected and analyzed.The longissimus dorsi muscle mass,posas minor muscle mass and each muscle mass in the hind leg were weighted.Additionally,fatty acid composition(%) of the diet,the longissimus dorsi muscle and the backfat were analyzed by gas chromatography method.PPARγand TNFαmRNA expression levels in muscle,spleen and adipose tissue,and plasma concentrations of TNFαdata were measured and analyzed.1.Duration of dietary linseed feeding affects the intramuscular fat,muscle mass and fatty acid composition in pig muscle.No significant difference(P＞0.05) was observed for average backfat thickness,lean meat percentage,loin muscle area, whereas the intramuscular fat content increased linearly(P＜0.01) as prolonged the time of feeding linseed diet.As prolonged the time of feeding linseed diet,the longissimus dorsi muscle mass,quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly(P＜0.01).Duration of feeding linseed diet linearly increased(P＜0.01) the LNA,EPA and C22:5n-3 concentrations in the longissimus dorsi muscle and backfat.There was significant quadratic relation between the intramuscular fat content and the n-3 polyunsaturated fatty acids(PUFA) enrichment (R~2=0.87,P＜0.01),or LNA enrichment(R~2=0.91,P＜0.01) in the longissimus dorsi muscle.Likewise,the longissimus dorsi muscle mass was also quadratically related to the n-3PUFA enrichment(R~2=0.89,P＜0.01),or LNA enrichment(R~2=0.86,P＜0.01) in the longissimus dorsi muscle.These results revealed that duration of feeding linseed diet may stimulate intramuscular fat accumulation,and promote the hypertrophy of the longissimus dorsi muscle,quadriceps femoris muscle and semitendinosus muscle mass by increasing the n-3PUFA enrichment,especially LNA enrichment in the longissimus dorsi muscle.2.Duration of feeding linseed diet influences peroxisome proliferator-activated receptorγand tumor necrosis factor gene expression,and muscle mass of growing-finishing barrows.The expression of PPARγin longissimus muscle and spleen increased(P＜0.01) linearly as prolonged the time of feeding linseed diet,while the expression of PPARγin adipose tissue were not affected(P=0.095).Duration of linseed addition linearly decreased(P＜0.01) TNFαgene expression levels in the longissimus dorsi muscle,adipose and spleen,and serum concentration of TNFαas well.The expression levels of PPARγnegatively correlated with the expression of TNFαin muscle(R~2=0.70,P＜0.001) and spleen(R~2=0.77,P＜0.001) respectively. Likewise,PPARγexpression level in spleen(R~2=0.59,P＜0.01) or muscle(R~2=0.52, P＜0.05) negative correlated with serum TNFαconcentration.There were significant quadratic relation between muscular PPARγ(R~2=0.80,P＜0.01) or muscular TNFα(R~2=0.87,P＜0.01) expression and the longissimus dorsi muscle mass.These results demonstrated that duration of feeding linseed diet lead to a linear decrease of TNFαgene expression,which may increase the muscle mass in growing-finishing barrows, at least in part,through a PPARγ-dependent mechanism.The second part:in vitro experiment.In order to investigate the molecular mechanism of n-3PUFA regulating skeletal muscle growth of growing-finishing pigs. C2C12 myotube were incubated with BSA(the postive control),PTA(the negative control),ALA an EPA,respectivley.After treatment with 600μM BSA,LNA,EPA or PTA for 24 hours PPARγ,TNFαand MuRF1 mRNA expression levels were measured by Real-time quantitative PCR method.After incubation of C2C12 myotubes with 300μM,600μM BSA,LNA,EPA or PTA for 24 hours,respectively,the abundance of IκBαwas measured by Western blot method.Additionally,C2C12 myotubes were incubated with 600μM LNA,600μM EPA,600μM PTA or for 24 h.Total nuclear protein was subsequently isolated and analyzed by EMSA for NF-κB DNA binding activity.The effect of n-3PUFA on NF-κB activation and MuRF1 gene expression in C2C12 skeletal muscle Cells.C2C12 myotubes incubated in the presence of 600μM EPA for 24 hours resulted in a 2.3-fold induction of the PPARγepression(P＜0.01).A 1.08-fold induction(P＜0.01) was observed in the presence of 600μM LNA for 24 hours.However,24-h incubation period with 600μM palmitate decreased PPARγexpression(P＜0.01).Treatment with 600μM LNA or EPA for 24 hours caused a 1.06-fold and 2.93-fold reduction in the mRNA levels of TNFαin C2C12 myotubes (P＜0.001),respectively.Whereas 24-h incubation period with 600μM palmitate increased TNFαexpression(P＜0.01).Incubation of C2C12 myotubes with 300μM LNA,EPA or palmitate for 24 hours did not affect the abundance of IκBα.However, 600μM EPA addition to cells caused approximately a 86%(P＜0.01) increase in the abundance of IκBα,and 600μM palmitate addition to cells caused approximately a 39%(P＜0.01) decrease in the abundance of IκBα.Incubation of C2C12 myotubes with 600μM EPA for 24 hours decreased the NF-κB DNA binding activity.However, Treatment with 600μM LNA or EPA for 24 hours increased the NF-κB DNA binding activity.C2C12 myotubes incubated in the presence of 600μM EPA for 24 hours caused a 3.38-fold induction in the levels of MuRF1 mRNA(P＜0.01).Whereas 24-h incubation period with 600μM palmitate increased MuRF1 expression(P＜0.01). These results revealed EPA treatment of skeletal muscle cells represses MuRF1 expression through mechanisms involving the activation of the axis PPARγ/TNFα/IκBα/NF-κB,decreases skeletal muscle protein degradation,and promotes the skeletal muscle growth.更多还原