Slc35d3对脂肪、肝脏、肾脏组织中脂代谢的影响

发布时间：2018-12-14 07:43

【摘要】：研究背景及目的肥胖是一种复杂的慢性疾病,也是21世纪代谢综合征最重要的表现之一。肥胖与心血管疾病的危险因素紧密相关,例如胰岛素抵抗、糖尿病、高血压和血脂异常等。肝脏是体内最大的脂肪酸合成及代谢器官,合成的脂肪酸会在载脂蛋白的协助下释放入血,在脂库中储存或为其他组织器官提供能量。脂肪组织是体内最大储能器官,多余的能量会以脂肪的形式储存,当能量不足时,发生脂肪动员,来满足机体所需。肝脏、心脏及肾脏等实质性器官很容易发生脂肪变性,其中的脂代谢非常关键。SLC35D3基因编码了一种核苷酸转运蛋白。已有相关文献报道Slc35d3是肥胖或代谢综合征的一个候选基因,其蛋白定位在内质网,与D1R相互作用。Slc35d3基因突变的小鼠会通过减弱纹状体神经元的多巴胺信号,从而导致代谢综合征,而且在两名代谢综合征的患者中发现了SLC35D3基因的突变。本实验主要是通过研究SLC35D3基因在脂肪、肝脏及肾脏组织中对脂代谢的影响。方法采用荧光定量PCR的方法检测16周龄的DIOB6J(饮食诱导的肥胖)小鼠、ob/ob(leptin缺陷)小鼠及正常对照C57BL/6J小鼠的脂肪、肝脏、肾脏组织中Slc35d3 mRNA转录水平是否变化;3T3-L1前脂肪细胞分化6天后电转染人源的SLC35D3基因,观察其对分化指标及脂代谢相关基因mRNA水平是否影响,同时检测是否影响葡萄糖消耗情况;同时在HepG2细胞中利用脂质体转染的方法过表达SLC35D3基因,转染两天后检测对脂代谢相关基因mRNA水平的影响;转后第3天用1000uM油酸、棕榈酸共同刺激24h,检测脂代谢相关基因mRNA水平的表达变化;刺激24h后恢复正常培养基,继续培养24h,并检测对脂代谢相关基因mRNA水平的影响;在293T细胞中利用脂质体的方法转染人源SLC35D3基因,转染2天后检测其对脂代谢相关基因转录水平的改变。结果在DIO B6J小鼠、ob/ob肥胖小鼠模型中,脂肪及肾脏组织中Slc35d3的转录水平明显低于C57BL/6J对照组(P0.01);在肥胖小鼠的肝脏组织中,Slc35d3的转录水平明显高于对照组(P0.01)。3T3-L1前脂肪细胞分化6天后过表达人源SLC35D3基因后,脂肪细胞分化指标基因及脂代谢相关基因mRNA表达显著降低,但是葡萄糖消耗差异不显著。HepG2细胞过表达人源SLC35D3基因后,脂代谢相关基因的mRNA表达也显著降低。293T细胞过表达人源SLC35D3基因后,脂代谢相关基因的mRNA表达也显著降低。结论SLC35D3通过下调脂肪细胞分化相关基因和脂代谢相关基因影响了脂肪、肝脏及肾脏组织中脂代谢过程,可作为将来研究肥胖或代谢综合征的候选基因。
[Abstract]:Background and objective Obesity is a complex chronic disease and one of the most important manifestations of metabolic syndrome in the 21st century. Obesity is closely associated with risk factors for cardiovascular disease, such as insulin resistance, diabetes, hypertension, and dyslipidemia. The liver is the largest fatty acid synthesis and metabolic organ in the body. The synthesized fatty acids are released into the blood with the help of apolipoprotein and stored in the lipid bank or provide energy for other tissues and organs. Adipose tissue is the largest energy storage organ in the body. The excess energy is stored in the form of fat. When the energy is insufficient, fat mobilization occurs to meet the needs of the body. Fatty degeneration is easy to occur in parenchymal organs such as liver, heart and kidney, in which lipid metabolism is crucial. SLC35D3 gene encodes a nucleotide transporter. It has been reported that Slc35d3 is a candidate gene for obesity or metabolic syndrome, whose protein is located in the endoplasmic reticulum and interacts with D1R. Mice with Slc35d3 gene mutation can attenuate dopamine signal in striatal neurons. This led to metabolic syndrome, and mutations in the SLC35D3 gene were found in two patients with metabolic syndrome. The purpose of this study was to investigate the effect of SLC35D3 gene on lipid metabolism in fat, liver and kidney tissues. Methods the changes of Slc35d3 mRNA transcription in fat, liver and kidney of DIOB6J (diet-induced obesity) mice, ob/ob (leptin deficient) mice and normal control C57BL/6J mice were detected by fluorescence quantitative PCR. Human SLC35D3 gene was electrotransfected into human adipocytes 6 days after differentiation of 3T3-L1. The effects of SLC35D3 gene on differentiation index and mRNA level of lipid metabolism related gene were observed. At the same time, glucose consumption was also detected. At the same time, the SLC35D3 gene was overexpressed in HepG2 cells by liposome transfection. After two days of transfection, the effect of liposome transfection on the mRNA level of lipid metabolism-related genes was detected. On the third day, 1000uM oleic acid and palmitic acid were used to detect the expression of lipid metabolism-related gene mRNA for 24 h, and the normal medium was restored to culture for 24 h after stimulation, and the effect on the mRNA level of lipid metabolism-related gene was detected. Human SLC35D3 gene was transfected into 293T cells by liposome. The changes of lipid metabolism-related gene transcription level were detected after transfection for 2 days. Results in DIO B6J mice and ob/ob obese mice, the transcription level of Slc35d3 in fat and kidney tissues was significantly lower than that in C57BL/6J control group (P0.01). In the liver tissue of obese mice, the transcription level of Slc35d3 was significantly higher than that of control group (P0.01). After 6 days of differentiation of 3T3-L1 preadipocytes, human SLC35D3 gene was overexpressed. The expression of adipocyte differentiation index gene and lipid metabolism-related gene mRNA was significantly decreased, but glucose consumption was not significantly different. HepG2 cells overexpressed human SLC35D3 gene. The mRNA expression of lipid metabolism-related genes was also significantly decreased, and the mRNA expression of lipid metabolism-related genes was also significantly decreased after 293T cells over-expressed the human SLC35D3 gene. Conclusion SLC35D3 can be used as a candidate gene for the study of obesity or metabolic syndrome because of its down-regulation of adipocyte differentiation related genes and lipid metabolism-related genes in adipose, liver and kidney tissues.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2

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