【摘要】：研究背景及目的肥胖是一種復(fù)雜的慢性疾病,也是21世紀(jì)代謝綜合征最重要的表現(xiàn)之一。肥胖與心血管疾病的危險(xiǎn)因素緊密相關(guān),例如胰島素抵抗、糖尿病、高血壓和血脂異常等。肝臟是體內(nèi)最大的脂肪酸合成及代謝器官,合成的脂肪酸會(huì)在載脂蛋白的協(xié)助下釋放入血,在脂庫(kù)中儲(chǔ)存或?yàn)槠渌M織器官提供能量。脂肪組織是體內(nèi)最大儲(chǔ)能器官,多余的能量會(huì)以脂肪的形式儲(chǔ)存,當(dāng)能量不足時(shí),發(fā)生脂肪動(dòng)員,來(lái)滿足機(jī)體所需。肝臟、心臟及腎臟等實(shí)質(zhì)性器官很容易發(fā)生脂肪變性,其中的脂代謝非常關(guān)鍵。SLC35D3基因編碼了一種核苷酸轉(zhuǎn)運(yùn)蛋白。已有相關(guān)文獻(xiàn)報(bào)道Slc35d3是肥胖或代謝綜合征的一個(gè)候選基因,其蛋白定位在內(nèi)質(zhì)網(wǎng),與D1R相互作用。Slc35d3基因突變的小鼠會(huì)通過(guò)減弱紋狀體神經(jīng)元的多巴胺信號(hào),從而導(dǎo)致代謝綜合征,而且在兩名代謝綜合征的患者中發(fā)現(xiàn)了SLC35D3基因的突變。本實(shí)驗(yàn)主要是通過(guò)研究SLC35D3基因在脂肪、肝臟及腎臟組織中對(duì)脂代謝的影響。方法采用熒光定量PCR的方法檢測(cè)16周齡的DIOB6J(飲食誘導(dǎo)的肥胖)小鼠、ob/ob(leptin缺陷)小鼠及正常對(duì)照C57BL/6J小鼠的脂肪、肝臟、腎臟組織中Slc35d3 mRNA轉(zhuǎn)錄水平是否變化;3T3-L1前脂肪細(xì)胞分化6天后電轉(zhuǎn)染人源的SLC35D3基因,觀察其對(duì)分化指標(biāo)及脂代謝相關(guān)基因mRNA水平是否影響,同時(shí)檢測(cè)是否影響葡萄糖消耗情況;同時(shí)在HepG2細(xì)胞中利用脂質(zhì)體轉(zhuǎn)染的方法過(guò)表達(dá)SLC35D3基因,轉(zhuǎn)染兩天后檢測(cè)對(duì)脂代謝相關(guān)基因mRNA水平的影響;轉(zhuǎn)后第3天用1000uM油酸、棕櫚酸共同刺激24h,檢測(cè)脂代謝相關(guān)基因mRNA水平的表達(dá)變化;刺激24h后恢復(fù)正常培養(yǎng)基,繼續(xù)培養(yǎng)24h,并檢測(cè)對(duì)脂代謝相關(guān)基因mRNA水平的影響;在293T細(xì)胞中利用脂質(zhì)體的方法轉(zhuǎn)染人源SLC35D3基因,轉(zhuǎn)染2天后檢測(cè)其對(duì)脂代謝相關(guān)基因轉(zhuǎn)錄水平的改變。結(jié)果在DIO B6J小鼠、ob/ob肥胖小鼠模型中,脂肪及腎臟組織中Slc35d3的轉(zhuǎn)錄水平明顯低于C57BL/6J對(duì)照組(P0.01);在肥胖小鼠的肝臟組織中,Slc35d3的轉(zhuǎn)錄水平明顯高于對(duì)照組(P0.01)。3T3-L1前脂肪細(xì)胞分化6天后過(guò)表達(dá)人源SLC35D3基因后,脂肪細(xì)胞分化指標(biāo)基因及脂代謝相關(guān)基因mRNA表達(dá)顯著降低,但是葡萄糖消耗差異不顯著。HepG2細(xì)胞過(guò)表達(dá)人源SLC35D3基因后,脂代謝相關(guān)基因的mRNA表達(dá)也顯著降低。293T細(xì)胞過(guò)表達(dá)人源SLC35D3基因后,脂代謝相關(guān)基因的mRNA表達(dá)也顯著降低。結(jié)論SLC35D3通過(guò)下調(diào)脂肪細(xì)胞分化相關(guān)基因和脂代謝相關(guān)基因影響了脂肪、肝臟及腎臟組織中脂代謝過(guò)程,可作為將來(lái)研究肥胖或代謝綜合征的候選基因。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.2

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