【作者】 高晓娟；

【导师】 杨公社；

【作者基本信息】 西北农林科技大学 ， 动物学， 2011， 硕士

【摘要】 脂肪组织是机体重要的能量调节和内分泌器官,脂肪沉积与家畜肉质改善及肥胖症、二型糖尿病、心血管疾病等人类代谢性疾病的发生密切相关,因此研究脂肪细胞分化在家畜遗传育种、人类代谢疾病治疗学等方面具有重要意义。然而,脂肪细胞分化是众多关键基因及相关信号通路参与的复杂的网络调控过程,PGC-1β作为脂肪细胞分化标记基因PPARγ的辅激活物,可通过辅激活众多转录因子而发挥作用,并发现PGC-1β与脂肪细胞分化密切相关,其基因敲除鼠表现为脂肪组织含量减少,但其在脂肪细胞分化中的作用机理尚不清楚。SREBP-1c作为另一个成脂重要的决定因子,在脂肪和肝脏成脂中起重要作用,且PGC-1β可以通过辅激活SREBP-1c而调节肝细胞的成脂,而在脂肪细胞中是否存在相似的成脂机理尚不清楚。本研究为了探讨以上问题,以1~3日龄仔猪为实验动物,利用Real-time PCR、Western Blot和细胞免疫荧光染色法分析PGC-1β和SREBP-1c在猪脂肪细胞分化过程中的表达模式。通过PGC-1β的慢病毒干扰载感染猪原代脂肪细胞,采用油红O染色法鉴定细胞内甘油三酯积累的变化;Real-time PCR方法分析PGC-1β、分化标志基因PPARγ和SREBP-1c及其下游靶基因FAS和ACC的mRNA表达变化;Western blot方法检测PGC-1β、、SREBP-1c和脂肪细胞分化关键转录因子C/EBPα的蛋白表达变化;免疫共沉淀方法分析PGC-1β与SREBP-1c蛋白的相互作用。最终明确PGC-1β在调控猪脂肪细胞分化中的作用,及其调节并结合SREBP-1c的机理,主要研究结果如下:1. PGC-1β和SREBP-1c在分化的猪脂肪细胞中高表达,并伴随分化关键基因PPARγ和C/EBPα的表达而在脂肪细胞分化过程中逐渐增加,且二者在分化细胞的核质均有分布,但主要分布于细胞质。2. PGC-1β的干扰显著下调了SREBP-1c的mRNA和蛋白表达,同时抑制了脂肪细胞分化标记基因PPARγ和C/EBPα的表达,并降低了脂肪细胞内甘油三脂的积累,从而抑制猪脂肪细胞分化。3.在分化的猪脂肪细胞中,PGC-1β和SREBP-1c存在蛋白水平的相互作用,可能与PGC-1β调节脂肪细胞分化相关。本实验旨在研究猪脂肪细胞分化过程中PGC-1β对SREBP-1c的调节和结合作用,初步证明了PGC-1β和SREBP-1c的表达与猪脂肪细胞分化密切相关,PGC-1β干扰抑制SREBP-1c表达及脂肪细胞细胞分化,同时,鉴定PGC-1β和SREBP-1c在分化的猪脂肪细胞中存在相互作用,从而为研究PGC-1β在脂肪细胞分化过程中的作用机理提供新途径。更多还原

【Abstract】 Adipose tissue as an important organ of energy regulation and endocrine, and the improvement of livestock fleshy and the human metabolic diseases such as obesity and type II diabetes and cardiovascular disease are all involved in fat deposit. So understanding adipocyte differentiation is of great significance for gaining insight into the livestock genetic breeding and pathogenesis of human metabolic diseases.Adipocyte differentiation is one complex network of regulatory processes, which involved in many key genes and signal pathways. PGC-1βas an activator of adipocyte differentiation markers PPARγ, it could play roles through coactivating a number of transcription factors. We have found that PGC-1βwas closely related to adipocyte differentiation, and the fat content of PGC-1βknockout mice was significantly reduced, but its mechanism in adipocyte differentiation is unclear currently. SREBP-1c as another important determinant factor of adipogenesis, played an important role in adipogenic of adipose and liver tissue. Moreover, we found that PGC-1βcould regulate adipogenesis by coactivating SREBP-1c in liver, but it is still unknown that whether there are similar adipogenic mechanisms in adipocytes.In order to discuss the problems mentioned above, 1-3 days piglets were used as our experimental animals. We detected the expression pattern of PGC-1βand SREBP-1c during porcine adipocyte differentiation by Real-time PCR, Western Blot and immunofluorescence method.Then, porcine primary adipocytes were infected with recombinant lentivirus interfering vector target on PGC-1β; Oil red O staining were used to determine the changes of triglyceride accumulation in cellular; the mRNA expression changes of PGC-1β, adipocyte differentiation markers PPARγ, SREBP-1c, its downstream target genes FAS and ACC were analyzed with Real-time PCR; The changes of PGC-1β, SREBP-1c, and adipocyte differentiation transcriptional factors C/EBPαproteins expression were analyzed with Western blot; Co-IP was used to investigate the interaction between PGC-1βand SREBP-1c proteins. In conclusion, we known the function of PGC-1βin regulating porcine adipocytes differentiation, and investigated the mechanism on modulating and interacting with SREBP-1c. The main results are as follows:1. PGC-1βand SREBP-1c are highly expressed in differentiated porcine adipocytes, and their expression gradually increased during adipocytes differentiation, accompanied with key genes PPARγand C/EBPαexpression. Besides, we observed that these two proteins located both in nucleus and cytoplasm in differentiated adipocytes, but main in cytoplasm.2. PGC-1βinterference significantly decreased the mRNA and protein expression of SREBP-1c, and inhibited adipocyte differentiation marker gene PPARγand C/EBPαexpression, the accumulation of triglyceride was also obviously reduced, so the differentiation of porcine adipocytes were inhibited.3. PGC-1βand SREBP-1c were interacted in protein levels in differentiated porcine adipocytes, it may be related to the mechanism of PGC-1βon regulating adipocyte differentiation.Intended to investigate the regulation and interaction of SREBP-1c on PGC-1βduring porcine preadipocytes differentiation, we preliminarily proved that the expression of PGC-1βand SREBP-1c were closely related to porcine adipocytes differentiation, interference of PGC-1βinhibited SREBP-1c expression and adipocyte differentiation. Furthermore, we identifinted the interaction between PGC-1βand SREBP-1c proteins in differentiated porcine adipocytes. It will provide a novel way to elucidate the function and mechanism of PGC-1βon modulating adipocyte differentiation.更多还原