【作者】 张辉；

【导师】 孙世铎； 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， 2009， 硕士

【摘要】 骨骼肌纤维类型根据其形态、功能和生理生化特性分为Ⅰ型和Ⅱ型,提高Ⅰ型肌纤维的比例是改善畜禽肉品质的主要途径。长期以来,国内外许多学者致力于通过功能基因调控肌纤维类型来改善畜禽肉品质。近年来研究表明,叉头转录因子O亚家族1(forkhead transcription factor group o1,FoxO1)可能是骨骼肌成肌细胞分化过程的重要调控因子,其通过多条通路在调控骨骼肌成肌细胞的增殖与分化中发挥重要作用。因此,本试验以猪为模式动物,利用细胞培养,sq RT-PCR,Western Blot,RNA干扰等细胞生物学和分子生物学技术,研究FoxO1在猪骨骼肌纤维类型发育过程中的调控作用。获得如下结果:1.在SOL、LD、EDL和GAST这四种类型骨骼肌中,mRNA水平检测发现,MyHCⅠ和MyHCⅡa的表达量在SOL中较高,而MyHCⅡb和MyHCⅡx在SOL中表达量较低;在EDL和GAST中,MyHCⅡb和MyHCⅡx的表达量较高。FoxO1在SOL和GAST中表达较高,但在LD和EDL中表达较低,FoxO1在这四种类型骨骼肌中的表达量与Sirt1和NFAT相似,但与Mef2c相反。Western Blot显示:FoxO1蛋白在SOL中表达最低。表明FoxO1在不同类型骨骼肌中表达量有差异,与肌纤维类型相关基因密切相关。2.体外培养诱导分化1~9d的骨骼肌成肌细胞发现,FoxO1基因mRNA的表达量变化不明显,但是其蛋白表达却随着细胞分化表达量逐渐上升,并且MyHCⅡb和MyHCⅡx的mRNA表达量也逐渐上升,与FoxO1蛋白表达趋势一致。表明在骨骼肌成肌细胞分化过程中,FoxO1与Ⅱ型肌纤维的表达相关。3.采用50nM的渥曼青霉素处理猪成肌细胞0,3,5天,利用sq RT- PCR和Western Blot等技术,检测肌肉形成与肌纤维类型相关基因的表达。结果显示:激活内源活性FoxO1, MyHCⅡb、MyHCⅡx和NFAT表达显著上调,而MyHCⅠ、MyHCⅡa和Mef2c表达显著下调。所以FoxO1可能通过NFAT和Mef2c调控肌纤维类型。4.根据RNAi引物设计原则,设计猪FoxO1基因shRNA的四条干扰引物序列,合成、退火形成双链寡核酸后克隆到慢病毒载体Lenti H1 vector的黏性末端,测序,初步成功连接FoxO1-shRNA慢病毒干扰载体,为进一步研究研究FoxO1基因在调节猪肌纤维类型转化过程中的作用机理奠定基础。更多还原

【Abstract】 Based on the different structural, functional, physics and chemical properties, Skeletal muscle is composed of typeⅠ,Ⅱa ,Ⅱx andⅡb. Through improving the content of typeⅠm uscle, we could make meat quality better. For a long period of time, many scholars want to improve meat quality by controling the content of muscle fiber type. The studies in recent years showed that FoxO1 might be the key regulatory factor during process of myoblast differentiation. It may influence cell proliferation and differentiation through multiple ways, hinting that procine FoxO1 plays an important modulatory role during formation of myocyte. Thereby, our study use pigs as model animal through cell culture, sq RT-PCR, Western Blot, RNAi and other methods in order to clarify the role of FoxO1 in the regulation of development of muscle fiber type. The main reserch results as follows:1. In the SOL, LD, EDL and GAST of skeletal muscle. RT-PCR detection showed the expression levels of MyHCⅠand MyHCⅡa were much higher, whereas the expression levels of MyHCⅡb and MyHCⅡx were much lower in SOL; In contrast, the expression levels of MyHCⅡb and MyHCⅡx were much higher in LD and EDL. The expression of FoxO1 were higher in SOL and GAST than in LD and EDL, in addition, the expression of FoxO1 in four types of skeletal muscle were similar to Sirt1 and NFAT, but opposite to Mef2c. Western Blot assay indicated FoxO1 protein is lowest in SOL. The results demonstrated that the expression of FoxO1 in four types of skeletal muscle were different, and closely related to muscle fiber type-related genes.2. We successfully separate and culture pig myocyte as a ideal material to study muscle fiber type.Through inducing differatiation in in vitro, we discovered during first 1 to 9 days, the content of FoxO1 protein increasd significantly but not the RNA level, with the time increasing, the level of MyHCⅡb and MyHCⅡx increased as the same as the he content of FoxO1 protein expression, however the RNA level of MyHCⅠand MyHCⅡdidn’t change. It is suggested during the differiation of myocyte, FoxO1 have an impaction on muscle fiber by poste-transcription.3. To study the role of the dephosphorylation of FoxO1 on muscle fiber type, we collected the skeletal muscle myoblast on the day0, day3 and day5 after being treated with Wortmannin. We detected the expression of FoxO1, skeletal muscle fiber type gene markers and genes involved in muscle fiber type regulation, such as MyHC I, MyHCⅡa, MyHCⅡb and MyHCⅡx, NFAT and Mef2c by RT-PCR and Western blot. Results: Activation of FoxO1 in porcine myoblast dramatically increased the expression of MyHCⅡb, MyHCⅡx and NFAT, while the expression of MyHCⅠ, MyHCⅡa and Mef2C decreased obviously. FoxO1 may regulate muscle fiber type through Mef2c and NFAT mediated pathway.4. RNAi design based on,the principle of primer design porcine FoxO1 gene shRNA sequence, synthesis, annealing after the formation of double-stranded oligonucleotides cloned into the lentiviral vector of the sticky end of Lenti H1 vector, sequencing, build FoxO1 gene RNAi lentiviral vector, bjective To construct a lentiviral vector-mediated RNAi of FoxO1 gene and provide the basis for further experiments in muscle fiber types.更多还原