【作者】 于太永；

【导师】 杨公社；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 现在越来越多的研究认为脂肪组织不只是传统意义上被动的储能器官,也是机体重要的内分泌器官。它可以产生和分泌多种脂肪源性激素或因子,不仅以中枢神经的方式参与机体能量代谢的调节,而且还可以自/旁分泌或内分泌的方式做用于其他部位,甚至远隔组织或器官,对外周组织的生长、发育和代谢等发挥广泛的调控作用。脂肪因子的调控失常将对人类及动物健康和生产产生重要影响。在过去几十年里,肥胖症及其相关疾病已经非常常见,在某些人群和社会流行速度直线上升。另一方面,随这人类消费水平的提高和消费观念的转变,降低动物体脂沉积和改善动物产品质量已是动物育种领域重要的研究课题。因此,认识脂肪因子的作用及其作用机制,从而为调控体脂沉积和改善动物产品质量在畜牧业生产中有着重要意义。脂肪和肌肉组织的生长和分化受到很多因子的调节。肌细胞和脂肪细胞间的相互作用对生长和发育起着重要作用,包括肌肉形成的速率和程度,肌肉的生长,脂肪的形成,脂肪的分解和能源物质的利用。与这一调节过程相关的重要激素和生长因子包括leptin、TNF-α、胰岛素样生长因子、各种细胞活素类和胰岛素等。其中leptin和TNF-α被认为在脂肪和细胞间的相互通讯中起着优为重要的作用。本研究以猪骨骼肌成肌细胞为研究对象,采用体外培养、四甲基偶氮唑蓝(MTT)法、流式细胞术、细胞免疫化学和反转录聚合酶链式反应(RT-RCR)等现代细胞和分子生物学技术与方法,研究外源性脂肪细胞因子leptin和TNFα对猪骨骼肌成肌细胞增殖与分化的影响,并对其可能的作用机制进行探讨。主要获得如下研究结果:1.在体外成功分离、纯化和培养了猪的骨骼肌成肌细胞,进行了传代培养与冻存。经系统的生物学性能检测,证明了所培养细胞能够表达骨骼肌成肌特征性转录因子(MyoD和MyoG)和蛋白(a-Sarcometric actin和myosin);冻存前后细胞形态和活力良好;染色体数目和核型正常,能够表达外源基因,遗传性能良好良好;无细菌、病毒和支原体污染。所分离培养的细胞可作为体外研究骨骼肌发育的理想模型。2.脂肪细胞因子leptin呈剂量和时间依赖性地促进猪骨骼肌成肌细胞的增殖,促进增殖期成肌细胞S+G2/M阶段细胞的积聚,减少G0/G1期细胞比例,诱导成肌细胞特异性增殖转录因子MyoD的表达;减少成肌细胞分化时期肌管融合,降低CK活性,抑制成肌细胞特异性分化转录因子MyoG的蛋白表达。以上leptin促进骨骼肌成肌细胞增殖、抑制其分化的生理作用受MAPK和ERK信号转导通路的调控。3.脂肪细胞因子TNFα抑制猪骨骼肌成肌细胞增殖并其促进其凋亡,对增殖期成肌细胞S+G2/M阶段细胞的积聚和成肌细胞特异性增殖转录因子MyoD的表达均有抑制效果;减少成肌细胞分化时期肌管融合,降低CK活性,抑制特征性蛋白MyHC的表达。TNFα以上作用可能是通过促进猪骨骼肌成肌细胞中SOCS3基因转录表达,或激活JNK、MAPK和ERK信号转导通路实现的。4.脂肪细胞因子TNFα抑制leptin对猪骨骼肌成肌细胞增殖和增殖期S+G2/M阶段细胞积聚的促进作用;降低leptin对猪骨骼肌成肌细胞分化期间对CK活性和MyoG表达的抑制效应。但TNFα对leptin促进猪骨骼肌成肌细胞增殖和抑制其分化的负性调控作用是可逆的。5.脂肪细胞因子leptin显著促进猪骨骼肌成肌细胞发育早期脂肪型脂肪酸结合蛋白(A-FABP)基因和骨骼肌慢肌肌浆球蛋白基因转录与翻译水平的表达;显著抑制骨骼肌快肌肌浆球蛋白基因mRNA和蛋白的表达。以上结果提示leptin可能参与猪骨骼肌成肌细胞生长早期阶段肉质嫩度的发育调控。综上所述,该研究结果在理论上阐明了leptin和TNF-α对猪骨骼肌成肌细胞增殖和分化的生理功能及其部分作用机制,丰富和拓展了对leptin和TNF-α生物学功能的认识,进一步为脂肪细胞内分泌功能的论断提供了新的证据;同时,在畜牧业生产上有望为肉用家畜的肉质改良奠定新的理论依据,提供新的思路和策略。更多还原

【Abstract】 Adipose tissue is not only a specialized passive storage organ of energy in original and classical view,but also an important endocrine secretion organ,which exerts a profound influence on growth,development,metabolism and health of mammalians through producing and secreting dozens of factors that act either in an auto/paracrine or endocrine fashion.The dysfunction of factors secretion by adipose tissue will lead to an critical influence in metabolism and health of human and animals.In the past several decades,obesity and obesity-related diseases have become extremely common,and tends to more and more severity among certain groups and community.With the boost of consumption level and the shift of consumption concept,it’s an important research goal to decrease fat accumulation and improve the meat quality in the domain of livestock breeding.So,it’s very important in zootechny to elucidate the function of factors secretion by adipose tissue,and molecular mechanism underlying these roles in control of fat accumulation and meat quality improved.The development of adipose tissue and muscle tissue is regulated by lots of factors, such as leptin,TNF-α,IGF-1,insulin and so on.The cross-talk between adipocyte and myocyte through the facors especially Leptin and TNF-α,is very important to their growth and development,including the rate and degree of muscle growth,adipogenesis,stearolysis and energy utilization.In the study,we investigate the effect and molecular mechanism of leptin and TNF-αon porcine myoblast proliferation and differentiation,through the way of primary cell cuture,MTT,flow cytometry assay,immuno-cytochemistry,RT-PCR and other molecular biology methods.The results are as follows:1.Primary porcine myoblast isolated from skeletal muscle,can be sub-cultured and cryopreservated.MyoD,MyoG,a-Sarcometric actin and myosin expressed in the cell,which are the markers of myoblast differentiated to myocyte;The porcine myoblast had fine genetic ability with normal morphalage,cell live,chromosome number and karyotype before and after cryopreservation,and without contamination by bacteria virus and Chlamydia.These results showed that myoblast we got can be serve as an ideal model for the study of skeltel muscle development.2.Leptin significantly promoted myoblast proliferation,MyoD expression and cell accumulation in the S+G2/M phase,in a dose-dependent manner.Furthermore,formation of myotubes and the myogenic index were markedly reduced,and creatine kinase(CK)activity, expressions of myogenin and myosin heavy chain(MyHC)protein were inhibited by leptin. Leptin promoted proliferation and inhibited differentiation in porcine skeletal myoblasts, which maybe regulated through the signal transduction of MAPK and ERK.3.TNF-αinhibited myoblast proliferation,induce apoptosis significantly,and decrease the MyOD expression and cell accumulation in the S+G2/M phase.Moreover,myotubes formation,creatine kinase(CK)activity,expressions of myosin heavy chain(MyHC)protein were inhibited by TNF-α.The effect of TNF-αon myoblast maybe regulated through inducing SOC-3 expression or activation the signal transduction of MAPK,ERK and JNK.4.The effect of leptin on promoting myoblast proliferation and cell accumulation in the S+G2/M phase were inhibited by TNF-α.Furthermore,the inhibition degree of leptin on CK activity and MyoG expression was weakened by TNF-α.But it is reversible that the influence of TNF-αon leptin’s effects to the myoblast proliferation and differentiation.5.leptin significantly induced the expression of A-FABP and slow-myosin and reduced that of fast-myosin at the early stage of myoblast development,which suggests that leptin takes part in the regulation of skeletal muscle tenderness at the early stage of porcine muscle development.All in concludes,this study not only elucidates the effect and molecular mechanism of leptin and TNF-αon porcine myoblast proliferation and differentiation,but also expands the known about biology function of leptin and TNF-α,which provides new proof to the assertion that adipocyte tissue is an important endocrine secretion organ,and helpful to improve livestock breeding.更多还原