基于一种新的天花板培养方法分析猪成熟脂肪细胞去分化过程中关键基因表达模式  

Analysis of Expression Patterns of Key Genes During the Pig(Sus scrofa) Mature Adipocyte Dedifferentiation Based on a Novel Ceiling Culture Model

作　　者：宋子仪[1] 史新娥[1] 杨浩[1] 高倩[1] 赵丽丽[1] 杨公社[1] 

机构地区：[1]西北农林科技大学动物科技学院,动物脂肪沉积与肌肉发育实验室,杨凌712100

出　　处：《农业生物技术学报》2013年第4期379-387,共9页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2011ZX08006-005);国家973计划项目(No.2012CB124705)

摘　　要：成熟脂肪细胞在天花板培养(ceiling culture)条件下可自发去分化为不含脂滴的成纤维细胞(dedifferentiated fat cell,DFAT),这种细胞体外可向多个谱系分化,因此近年来备受人们关注。为研究猪成熟脂肪细胞去分化过程中关键基因表达规律,本研究先分离、鉴定了猪成熟脂肪细胞,然后采用一种新的天花板培养法使其发生去分化,再用实时定量PCR(RT-qPCR)技术分析了猪成熟脂肪细胞去分化过程中关键基因的表达变化,最后用1μmol/L罗格列酮(Rosiglitazone)处理猪成熟脂肪细胞,研究增强成脂对去分化的影响。结果显示:分离的猪成熟脂肪细胞的纯度高达98.7%,并且新式天花板培养法能够高效的使其发生去分化;在去分化的过程中中后期成脂关键基因过氧化物酶体增殖物激活受体γ(PPARγ)、脂肪细胞型脂肪酸结合蛋白(aP2)和脂蛋白酯酶(LPL)的mRNA水平分别上调了8、3和7.5倍,而脂肪分解关键基因激素敏感脂酶(HSL)和脂肪组织甘油三酯脂肪酶(ATGL)的mRNA水平分别上调了40倍和10倍;罗格列酮处理能够显著抑制猪成熟脂肪细胞去分化(P<0.05)。这些结果说明,成熟脂肪细胞去分化是一个以脂解为主并伴有一定水平成脂的脂代谢过程,这为进一步研究成熟脂肪细胞去分化机理提供了理论依据。When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat （DFAT） cells. Because of those cells possessing multilineage differentiation potential, dedifferentiation has become a significant research topic in recent years. Here we firstly isolated pig mature adipocytes and indentified them by GENMED staining.Then, in order to clarify the mechanism of mature adipocyte dedifferentiation, a novel ceiling culture model was developed and the mRNA expression of marker genes during dedifferentiation couse was analysized using Real-time PCR. Finally, we assessed the effect of 1 μmol/L rosiglitazone on mature adipocyte dedifferentiation. The results showed that 98.7% of isolated cells were mononuclear mature adipocytes and these adipocytes could efficiently dedifferentiate into DFAT cells under a novel ceiling culture model. During the course of dedifferentiation, the mRNA levels of adipogenic marker peroxisome proliferator activated receptor 7 （PPART）, adipocyte-type fatty acid-binding protein （aP2） and lipoprotein lipase （LPL） were up-regulated by 8, 3 and 7.5 folds, respectively. While the lipolytic marker hormone-sensitive lipase （HSL） and Adipose triglyceride lipase （ATGL） were increased about 40 and 10 folds in mRNA level, respectively. In addition, the dedifferentiation was remarkably suppressed by treatment with 1 μmol/mL rosiglitazone. These results indicate that it is mainly a lipolytic course companying with little ability of adipogenesis that mature adipocytes dedifferentiate into DFAT cells, and this will provide new theoretical reference for future research.

关 键 词：猪成熟脂肪细胞 新式天花板培养法 去分化 脂解 

分 类 号：S828.1[农业科学—畜牧学]