【摘要】：目的:體內(nèi)存在過多的糖皮質(zhì)激素進(jìn)而導(dǎo)致機(jī)體代謝出現(xiàn)紊亂。針對(duì)不同組織中糖皮質(zhì)激素受體與代謝相關(guān)的通路研究是治療慢性代謝疾病的發(fā)展的新靶點(diǎn)。已有研究證實(shí)成人體內(nèi)有代謝活性的棕色脂肪細(xì)胞。長(zhǎng)時(shí)間給予b-腎上腺素能受體激動(dòng)劑刺激后,小鼠體內(nèi)的白色脂肪細(xì)胞向棕色脂肪細(xì)胞轉(zhuǎn)化增加產(chǎn)熱的過程又稱“白色脂肪棕色化”。目前隨著研究的進(jìn)展,增強(qiáng)脂肪組織棕色化功能已成為治療機(jī)體代謝紊亂的新靶點(diǎn)。microRNA在多種生物學(xué)進(jìn)程中有至關(guān)重要的調(diào)控作用,具有高度的組織特異性。本研究從糖皮質(zhì)激素影響脂肪棕色化功能入手,篩選出特異的microRNA,研究糖皮質(zhì)激素調(diào)控白色脂肪棕色化及棕色脂肪功能的具體分子機(jī)制。方法:體內(nèi)研究方面,我們使用有活性的糖皮質(zhì)激素-地塞米松腹腔注射C57/B6小鼠6周,進(jìn)一步分析糖皮質(zhì)激素對(duì)小鼠白色脂肪形態(tài)、棕色化功能基因表達(dá)的影響。體外研究方面,使用不同濃度的地塞米松干預(yù)原代白色及棕色脂肪細(xì)胞,分析糖皮質(zhì)激素對(duì)白色脂肪棕色化及棕色脂肪功能的影響;在細(xì)胞功能方面,檢測(cè)地塞米松對(duì)白色脂肪細(xì)胞氧耗率及棕色脂肪氧耗率的影響。結(jié)果:課題組前期研究發(fā)現(xiàn)地塞米松對(duì)白色脂肪棕色化以及棕色脂肪功能均有有抑制作用。篩選并制備人皮下脂肪組織及網(wǎng)膜脂肪組織microRNA芯片,分析后發(fā)現(xiàn)在皮下脂肪細(xì)胞及網(wǎng)膜脂肪細(xì)胞中地塞米松均明顯促進(jìn)mi R-19b的表達(dá)。進(jìn)一步分析miR-19b的基因序列發(fā)現(xiàn)其上游啟動(dòng)子區(qū)域存在糖皮質(zhì)激素反應(yīng)元件,說明糖皮質(zhì)激素可直接通過作用于miR-19b上游啟動(dòng)子區(qū)域的GRE調(diào)控其表達(dá)。miR-19b與UCP1mRNA在不同濃度地塞米松刺激下的表達(dá)趨勢(shì)呈負(fù)相關(guān)且存在地塞米松濃度依賴性。高表達(dá)mi R-19b可抑制調(diào)節(jié)白色脂肪棕色化,減低細(xì)胞氧耗率。干涉miR-19b后細(xì)胞的棕色化的能力明顯加強(qiáng),氧耗顯著增加,而經(jīng)過地塞米松刺激后鈍化了miR-19b干涉對(duì)脂肪細(xì)胞棕色化功能的影響。實(shí)驗(yàn)亦發(fā)現(xiàn)miR-19b抑制棕色脂肪功能且再次證實(shí)地塞米松可抑制棕色脂肪功能。預(yù)測(cè)并證實(shí)ADRB1(Adrenergic Receptor b1)是miR-19b的靶基因且ADRB1可以促進(jìn)白色脂肪棕色化及棕色脂肪功能。結(jié)論:糖皮質(zhì)激素及其受體通過與miR-19b上游啟動(dòng)子序列中的GRE結(jié)合促進(jìn)miR-19b生成,進(jìn)一步作用于靶基因ADRB1,通過抑制其表達(dá)限制了白色脂肪細(xì)胞棕色化能力及棕色脂肪細(xì)胞功能,導(dǎo)致機(jī)體能耗減低、脂肪蓄積引起肥胖,最終引起胰島素抵抗、二型糖尿病等代謝紊亂。
[Abstract]:Objective: the presence of excessive glucocorticoids in the body leads to metabolic disorders. The study of glucocorticoid receptors and metabolic pathways in different tissues is a new target for the treatment of chronic metabolic diseases. Studies have shown that brown fat cells are metabolically active in adults. After stimulation with badrenergic receptor agonists for a long time, the transformation of white adipocytes into brown adipocytes in mice was also known as "white fat browning". With the development of research, enhancing the brown function of adipose tissue has become a new target for the treatment of metabolic disorders. MicroRNA plays a crucial role in many biological processes and has a high tissue specificity. In this study, the effects of glucocorticoids on the function of fat browning were studied, and the specific molecular mechanism of glucocorticoid regulating white fat browning and brown fat function was screened out by specific microRNA,. Methods: in vivo, we injected C57/B6 mice with active glucocorticoid-dexamethasone for 6 weeks, and further analyzed the effect of glucocorticoid on the expression of white fat and brown functional genes in mice. In vitro, different concentrations of dexamethasone were used to interfere with primary white and brown adipocytes, and the effects of glucocorticoid on the browning of white fat and the function of brown fat were analyzed. The effects of dexamethasone on the oxygen consumption rate and brown fat oxygen consumption rate of white adipocytes were measured. Results: the results showed that dexamethasone inhibited the browning of white fat and the function of brown fat. The microRNA microarray of human subcutaneous adipose tissue and omental adipose tissue was screened and prepared. It was found that dexamethasone significantly promoted the expression of mi R-19b in subcutaneous adipocytes and omental adipocytes. Further analysis of the gene sequence of miR-19b revealed that there were glucocorticoid response elements in the upstream promoter region of miR-19b. The results indicated that glucocorticoid could directly regulate the expression of glucocorticoid through GRE acting on the upstream promoter of miR-19b. There was a negative correlation between miR-19b and UCP1mRNA stimulated by different concentrations of dexamethasone, and there was a concentration dependence of dexamethasone on the expression of glucocorticoid. Overexpression of mi R-19b inhibited the regulation of white fat browning and reduced cell oxygen consumption. After interfering with miR-19b, the ability of browning of adipocytes was obviously enhanced, and the oxygen consumption was significantly increased, while the effect of miR-19b interference on the brown function of adipocytes was passivated after stimulation of dexamethasone. It was also found that miR-19b inhibited the function of brown fat and confirmed that dexamethasone could inhibit the function of brown fat. ADRB1 (Adrenergic Receptor b1) is a target gene of miR-19b and ADRB1 can promote the function of white fat brown and brown fat. Conclusion: glucocorticoid and its receptor promote miR-19b production by binding to GRE in upstream promoter sequence of miR-19b, and further act on target gene ADRB1,. Inhibition of its expression limits the brown ability of white adipocytes and the function of brown adipocytes, resulting in decreased energy consumption, fat accumulation, obesity, insulin resistance, type 2 diabetes mellitus and other metabolic disorders.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R589

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