糖皮质激素通过miR-19b调控脂肪棕色化功能的研究
发布时间：2019-01-08 11:45
【摘要】：目的:体内存在过多的糖皮质激素进而导致机体代谢出现紊乱。针对不同组织中糖皮质激素受体与代谢相关的通路研究是治疗慢性代谢疾病的发展的新靶点。已有研究证实成人体内有代谢活性的棕色脂肪细胞。长时间给予b-肾上腺素能受体激动剂刺激后,小鼠体内的白色脂肪细胞向棕色脂肪细胞转化增加产热的过程又称“白色脂肪棕色化”。目前随着研究的进展,增强脂肪组织棕色化功能已成为治疗机体代谢紊乱的新靶点。microRNA在多种生物学进程中有至关重要的调控作用,具有高度的组织特异性。本研究从糖皮质激素影响脂肪棕色化功能入手,筛选出特异的microRNA,研究糖皮质激素调控白色脂肪棕色化及棕色脂肪功能的具体分子机制。方法:体内研究方面,我们使用有活性的糖皮质激素-地塞米松腹腔注射C57/B6小鼠6周,进一步分析糖皮质激素对小鼠白色脂肪形态、棕色化功能基因表达的影响。体外研究方面,使用不同浓度的地塞米松干预原代白色及棕色脂肪细胞,分析糖皮质激素对白色脂肪棕色化及棕色脂肪功能的影响;在细胞功能方面,检测地塞米松对白色脂肪细胞氧耗率及棕色脂肪氧耗率的影响。结果:课题组前期研究发现地塞米松对白色脂肪棕色化以及棕色脂肪功能均有有抑制作用。筛选并制备人皮下脂肪组织及网膜脂肪组织microRNA芯片,分析后发现在皮下脂肪细胞及网膜脂肪细胞中地塞米松均明显促进mi R-19b的表达。进一步分析miR-19b的基因序列发现其上游启动子区域存在糖皮质激素反应元件,说明糖皮质激素可直接通过作用于miR-19b上游启动子区域的GRE调控其表达。miR-19b与UCP1mRNA在不同浓度地塞米松刺激下的表达趋势呈负相关且存在地塞米松浓度依赖性。高表达mi R-19b可抑制调节白色脂肪棕色化,减低细胞氧耗率。干涉miR-19b后细胞的棕色化的能力明显加强,氧耗显著增加,而经过地塞米松刺激后钝化了miR-19b干涉对脂肪细胞棕色化功能的影响。实验亦发现miR-19b抑制棕色脂肪功能且再次证实地塞米松可抑制棕色脂肪功能。预测并证实ADRB1(Adrenergic Receptor b1)是miR-19b的靶基因且ADRB1可以促进白色脂肪棕色化及棕色脂肪功能。结论:糖皮质激素及其受体通过与miR-19b上游启动子序列中的GRE结合促进miR-19b生成,进一步作用于靶基因ADRB1,通过抑制其表达限制了白色脂肪细胞棕色化能力及棕色脂肪细胞功能,导致机体能耗减低、脂肪蓄积引起肥胖,最终引起胰岛素抵抗、二型糖尿病等代谢紊乱。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589
本文编号：2404554
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589
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