【作者】 王虹；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 牛肉作为一种高蛋白、低胆固醇的肉类食品倍受人们青睐,其嫩度和风味可随着肌内脂肪含量的增加而得到改善。而动物脂肪的形成与沉积是受多种转录因子调控的复杂生理过程,与脂肪细胞的增殖分化密切相关。CCAAT增强子结合蛋白家族(CCAAT/enhancer-binding protein family, C/EBPs)是一类非常重要的转录因子,在脂肪细胞的增殖分化过程中起关键调控作用。目前,关于C/EBPs对牛成纤维细胞成脂转分化的影响及其调控机理仍不清楚。为了探索牛C/EBPs基因的功能,本研究以C/EBPs家族中与脂肪细胞增殖分化有着紧密联系的3个成员——C/EBPα、C/EBPβ和C/EBP8为目标基因,以牛成纤维细胞为实验对象,以腺病毒介导的基因过量表达技术为手段,直接发掘3个目标基因诱导牛成纤维细胞转分化为脂肪细胞的潜在能力。同时,在转录水平上初步探索目标基因之间的相互调控作用,及其对一些重要的脂肪细胞分化相关基因的影响。主要研究成果如下：(1)成功克隆得到秦川牛C/EBPα、C/EBPβ和C/EBPδ基因的CDS区序列,其长度依次为1062bp、1047bp和771bp,分别编码353、348和256个氨基酸残基。序列信息分析结果显示,牛C/EBPs与人相应基因的氨基酸序列同源性分别达95%、97%和87%；牛C/EBPα和C/EBPβ分别在第233-252位,第190-208位氨基酸区域存在由外向内的显著跨膜结构,而C/EBPδ则在第125-141位氨基酸区域存在反方向的跨膜结构；牛C/EBPα、C/EBPβ和C/EBPδ都含一个有完全由α-螺旋构成的亮氨酸拉链,符合C/EBPs家族的一般结构特征。(2)构建了牛C/EBPα、C/EBPβ和C/EBPδ基因的腺病毒过表达载体,并包装获得了高滴度重组腺病毒,建立了适合于牛成纤维细胞的腺病毒过表达载体系统。(3)成功诱导了牛成纤维细胞向脂肪细胞的转分化。油红O染色结果显示,分别单一过量表达牛C/EBPα、C/EBPβ或C/EBPδ基因,可诱导成纤维细胞转分化为脂肪细胞或脂肪样细胞。同时过量表达2个或3个目标基因,也可诱导成纤维细胞转分化为脂肪细胞,且细胞的表型变化比单一表达时更明显。(4) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可互相激活对方的转录表达,而都对C/EBPδ基因的表达无明显影响；过量表达C/EBPδ基因可在诱导细胞分化早期上调C/EBPα基因的mRNA表达量,而对C/EBPβ基因的表达无明显影响。同时过量表达C/EBPβ和C/EBPδ基因可在诱导细胞分化中期上调C/EBPβ基因的表达量。(5) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可在诱导细胞分化后期,上调过氧化物酶体增殖激活受体-gamma (PPARγ)基因的mRNA表达量,而过量表达C/EBPδ基因对PPARγ基因的表达无明显影响；同时过量表达C/EBPβ和C/EBPα、C/EBPβ和C/EBPδ基因,也可在细胞分化后期上调PPARγ基因的表达量。单一或同时过量表达目标基因均对固醇调节元件结合蛋白-1(SREBP1)基因的表达无明显影响。(6) Real-time PCR分析结果显示,过量表达C/EBPα或C/EBPβ基因可上调脂肪细胞分化相关基因的mRNA表达量,如脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶-α(ACCα)、脂肪酸合成酶(FAS)、硬脂酰辅酶A去饱和酶(SCD)、脂肪酸转运蛋白-1(FATP1)和补体D (CFD)基因；而过量表达C/EBP8基因则仅可上调LPL、CFD和脂肪酸结合蛋白-4(FABP4)基因的表达量。同时过量表达2个或3个目标基因,均可上调除FATP1和FABP4以外的其他上述基因的表达量。(7)FABP4基因的mRNA表达量仅在单一过量表达C/EBPδ基因的细胞组中显著上升,而在过量表达C/EBPβ基因的细胞组中还有一定程度的下降,表明过量表达牛C/EBPβ或C/EBPδ基因,对FABP4基因在转录水平上的调控作用可能有所差异。综上所述,本研究利用腺病毒过表达技术介导牛C/EBPα、C/EBPβ和C/EBPδ基因过量表达,首次成功诱导牛成纤维细胞转分化为脂肪细胞或脂肪样细胞。同时,在转录水平上初步探索了这3个目标基因之间的相互作用关系,及其对一些重要的脂肪细胞分化相关基因的调控作用,为进一步研究牛体内脂肪的形成与沉积提供一定的理论依据,为下一步培育转基因牛的相关科研工作奠定基础。更多还原

【Abstract】 Beef is considered as one of the healthy foods for human because of its abundant nutrient. Its flavor and tenderness can be improved by inducing intramuscular fat composition. Previous studies in vitro and in vivo on various kinds of animals elucidated that adipocytes proliferation and differentiation at cellular level were regulated by some important transcription factors. CCAAT-enhancer-binding protein family (C/EBPs) as one of these transcription factor families played pivotal role in the control of fat formation as well as fatty acids metabolism. However, there is little information concerning the function of bovine C/EBPα, C/EBPβ and C/EBPδ genes. Herein, we detected the potential specific effects of the three genes and their possible interactions during bovine fibroblaststrans-differentiation process. Furthermore, we also investigated their effects on several other fat cell differentiation related genes. The main results were as follows:(1) The complete CDS sequences of bovine C/EBPα, C/EBPβ and C/EBPδ genes were successfully obtained. The CDS areas of bovine C/EBPα, C/EBPβ and C/EBPδ genes were1062bp,1047bp and771bp in length, coding353,348,256amino acid residues respectively. The putative amino acid sequences showed bovine C/EBPα, C/EBPβ and C/EBPδ shared higher similarities of95%,97%and87%with their homologue respectively. Bovine C/EBPα and C/EBPβ occupied one outside to inside transmembrane domain, locating within amino acid from233to252and from190to208respectively, while C/EBPδ occupied one inside to outside transmembrane domain, locating within amino acid from125to141. Besides, all the three putative proteins had one Basic leucine zipper domain (bZIP) consisted of a helixes, consistent with the predominant characteristic of C/EBPs.(2) Recombinant adenovirus carrying bovine C/EBPα, C/EBPβ and C/EBPδ complete CDS fragments were successfully constructed, and further packaged and amplified in HEK293A cell lines.(3) Oil O staining results demonstrated that over-expression of C/EBPα, C/EBPβ or C/EBPδ converted bovine fibroblasts into adipocytes or adipocytes-like cells. Over-expression of the combination of the two or the three isoforms converted fibroblasts into fat-laden adipocytes. (4) Real-time PCR results indicated over-expression of C/EBPα or C/EBPβ enhanced the mRNA expression of each other during bovine fibroblasts trans-differentiation, while none of them affected C/EBPδ mRNA expression. Meanwhile, simultaneous up-regulation of C/EBPβ and C/EBP5enhanced C/EBPa mRNA expression at some level.(5) Real-time PCR results indicated over-expression of C/EBPα and/or C/EBPβ upregulated PPARy mRNA expression level in the late phase of cell differentiation, while C/EBP8did not. All the three genes had no significant influence on SREBP1gene.(6) Real-time PCR results indicated over-expression C/EBPα or C/EBPβ upregulated the mRNA expression level of genes related to adipocytes such as Lipoprotein lipase (LPL), Acetyl-CoA carboxylase alpha (ACC-a), Fatty acid synthase (FAS), Stearoyl-CoA desaturase (SCD), Fatty acid transporter member1(FATP1) and Complement Factor D (Adipsin/CFD) genes. Over-expression of C/EBPδ increased the mRNA expression level of LPL, CFD and Fatty acid binding protein4(FABP4) genes. Combination of C/EBPα, C/EBPβ and C/EBPδ over-expression enhanced the expression of the above mentioned genes except FABP4and FATP1.(7) The mRNA level of FABP4gene significantly increased only in cells in which C/EBP8was upregulated, on the contrary, it decreased when C/EBPβ was upregulated to some extent, implying the two C/EBP isoforms may have dissimilar effects on FABP4gene at cellular level in bovine.In conclusion, we successfully converted bovine fibroblasts into adipocytes or adipocytes-like cells by over-expressing bovine transcription factors namely C/EBPα, C/EBPβ and C/EBPδ, either individually or by combination. Also a simple sketch map between the three transcription factors interaction, and their effects on other related genes was obtained based on the present results.更多还原