增强血管周围棕色脂肪细胞功能对血管保护作用的机制研究

发布时间：2018-06-03 23:06

本文选题：棕色脂肪组织 + 血管周围脂肪组织　；参考：《第二军医大学》2016年博士论文

【摘要】：背景:全球范围内以动脉粥样硬化为病理基础的心脑血管疾病逐年增多,已经成为危害人类健康的首位疾病。普遍认为动脉粥样硬化病变始于内膜损伤诱发的炎症细胞浸润及脂质沉积,整个过程是“由内而外”的。但是,越来越多的证据显示始于外膜的病变也可导致动脉粥样硬化斑块的形成,其发生发展也可是“由外而内”的。体内大多数动脉外层均有脂肪包裹,称之为血管周围脂肪。既往认为脂肪组织的主要功能是保温及机械保护,深入的研究发现脂肪组织是体内重要的内分泌器官,调节多种生理功能。血管周围脂肪与其他部位脂肪组织相似可以通过内分泌及旁分泌众多细胞因子或者脂肪因子直接调节血管功能。人体内脂肪主要分为白色脂肪及棕色脂肪,活化棕色脂肪可改善代谢功能,降低血糖及血脂,抑制肥胖。近年来的研究已经较为明确的阐明了棕色脂肪功能细胞活化及分化调节的相关机制,使得活化棕色脂肪功能治疗疾病成为可能。目的:通过β3受体激动剂及转基因方法(PRDM16)诱导血管周围脂肪组织中棕色脂肪功能活化,观察棕色脂肪功能活化对血管舒缩功能及动脉粥样硬化病变的影响。转染PRDM16诱导脂肪前体细胞分化成为棕色脂肪细,观察对内皮细胞及巨噬细胞的功能的影响。观察棕色脂肪调控基因PGC-1α对内皮功能的作用。方法:C57BL/6J小鼠随机分成三组,分别予以正常饮食,高脂饮食以及高脂喂养+腹主动脉周围脂肪组织中CL-316243注射组。Apo E-/-小鼠随机分成腹主动脉周围脂肪组织假手术处理(生理盐水),空腺病毒载体转染以及PRDM16腺病毒载体转染。每周称重,实验终点时留取血标本测定血脂及血糖。HE染色观察主动脉斑块及周围脂肪组织。通过腹主动脉环线性肌动描记测定离体血管收缩及舒张功能。RT-PCR测定脂肪分泌因子如:瘦素、脂联素、MCP-1、IL-8、TNF-α、IL-6以及IL-10。在3T3-L1细胞中过表达PRDM16诱导分化成为棕色脂肪细胞并与内皮细胞及巨噬细胞共培养。测定内皮细胞NO,eNOS,ET-1水平以及巨噬细胞表面LOX-1及CD36水平,同时测定巨噬细胞M1及M2极化状态。人主动脉内皮细胞转染PGC-1α后观察血管紧张素Ⅱ处理对于NO生成的影响,以及对内皮eNOS,PI3K以及Akt蛋白和mRNA水平的调控。结果:相对于正常饮食,高脂饮食显著增加小鼠体重,提高血脂血糖及血压水平,增加腹主动脉周围脂肪负荷,促使瘦素及炎症因子水平增高,脂联素水平降低。CL-316243腹主动脉周围注射未能显著改变小鼠代谢指标,也未能抑制脂肪组织炎症状态及改善内分泌功能。但是PVAT可诱导脂肪细胞向棕色脂肪样细胞分化,棕色脂肪基因UCP-1及线粒体水平增高。与PRDM16组相比,对照组及空病毒组腹主动脉斑块显著增多。PRDM16组腹主动脉PVAT中以棕色脂肪样细胞为主,而其他两组以白色脂肪细胞为主。PRDM16组PE或者KCl介导的腹主动脉血管环收缩显著减弱,而ACh介导的血管舒张增强。PRDM16转染后显著增高瘦素、脂联素以及IL-8 mRNA水平,降低MCP-1,TNF-α,IL-6以及IL-10mRNA水平。PRDM16转染3T3-L1细胞可诱导细胞向棕色脂肪细胞分化。将巨噬细胞与PRDM16诱导分化的棕色脂肪细胞共培养,巨噬细胞表面LOX-1和CD36水平下降,此外巨噬细胞内MCP-1,IL-6及TNF-α下降,而IL-10增高。将人主动脉内皮细胞与AngⅡ共孵育后NO生成及PGC-1α表达水平均下调,过表达PGC-1α后内皮细胞内cGMP及亚硝酸盐含量水平增高,提示对AngⅡ降低NO生成的抑制作用。PGC-1α可阻断AngⅡ介导的eNOS,PI3K以及Akt的去磷酸化。结论:高脂饮食可导致血管周围脂肪功能异常,并促进血管收缩及斑块形成。转染PRDM16可诱导腹主动脉周围脂肪中棕色脂肪功能活化,抑制斑块生长并保护血管舒缩功能。3T3-L1细胞表达PRDM16后可分化为棕色脂肪细胞,并抑制巨噬细胞的炎症状态。内皮细胞过表达PGC-1α通过PI3K-Akt途径抑制eNOS的去磷酸化水平,发挥其保护内皮功能的作用。
[Abstract]:Background: cardiovascular and cerebrovascular diseases, which are based on the pathological basis of atherosclerosis, have increased year by year in the world. It has become the first disease that endangers human health. It is generally believed that atherosclerotic lesions begin with infiltration of inflammatory cells and lipid deposition induced by intimal injury. The whole process is "from inside out". But more and more evidence is found. It is shown that lesions that start from the outer membrane can also cause atherosclerotic plaque formation, and its development is "out of the outside". Most of the outer layers of the body are fat wrapped, called perivascular fat. The main function of the fat tissue is that the main function of the adipose tissue is the preservation of the heat and the protection of the machinery, and the fat tissue is found in depth. An important endocrine organ, which regulates a variety of physiological functions. The perivascular fat is similar to other parts of the adipose tissue, which can be directly regulated by endocrine and paracrine multiple cytokines or adipose factors. The body fat is mainly divided into white fat and brown fat, and the activation of brown fat can improve metabolic function and decrease. Blood sugar and blood lipid inhibit obesity. Recent studies have clearly elucidated the mechanism of activation and differentiation of brown fat functional cells, making it possible to activate brown fat function treatment. Objective: to induce brown fat work in the perivascular adipose tissue by the beta 3 receptor agonist and PRDM16 method. Can be activated to observe the effect of brown fat function activation on vasoconstriction and atherosclerotic lesions. Transfection of PRDM16 induced fat precursor cells into brown fat, the effect of observation on the function of endothelial cells and macrophages. Observe the effect of brown fat regulating gene PGC-1 alpha on endothelial function. Methods: C57BL/6J small Rats were randomly divided into three groups: normal diet, high fat diet and high fat feeding and abdominal aorta CL-316243 injection group.Apo E-/- mice randomly divided into abdominal aorta adipose tissue pseudo operation treatment (physiological saline), empty adenovirus vector transfection and PRDM16 adenovirus vector transfection. The blood samples were collected to determine the aortic plaque and the surrounding adipose tissue by.HE staining. The vasoconstrictor and diastolic function of the isolated aorta were measured by the abdominal aorta ring linear tracing and.RT-PCR was used to determine the fat secreting factors such as leptin, adiponectin, MCP-1, IL-8, TNF- a, IL-6, and IL-10. in 3T3-L1 cells. It became brown adipocytes and co cultured with endothelial cells and macrophages. The levels of NO, eNOS, ET-1, LOX-1 and CD36 on the surface of macrophages were measured, and the M1 and M2 polarization of macrophages were measured. The effect of tight Zhang Su treatment on the formation of NO in human aortic endothelial cells was observed after transfection of PGC-1 a, and the inside of the endothelial cells was observed. The regulation of skin eNOS, PI3K and Akt protein and mRNA levels. Results: compared with normal diet, high fat diet significantly increased the weight of mice, increased blood lipid glycemic and blood pressure levels, increased the fat load around the abdominal aorta, increased the levels of leptin and inflammatory factors, and decreased the level of adiponectin in the.CL-316243 abdominal aorta. The metabolic index of mice could not inhibit the inflammatory state of adipose tissue and improve the endocrine function. However, PVAT could induce the differentiation of fat cells to brown adipose cells, the increase of the UCP-1 and mitochondria of the brown fat gene. Compared with the PRDM16 group, the abdominal aorta in the control group and the empty virus group increased the abdominal aorta in PVAT of the.PRDM16 group. Brown adipose like cells were dominant, while the other two groups with white adipocytes as the main.PRDM16 group PE or KCl mediated the contraction of the abdominal aorta, while ACh mediated vasodilatation enhanced.PRDM16 transfection significantly increased leptin, adiponectin and IL-8 mRNA levels, reduced MCP-1, TNF- a, IL-6, and IL-10mRNA.PRDM16. 3T3-L1 cells could induce cells to differentiate into brown adipocytes. The macrophage and PRDM16 induced brown adipocytes were co cultured, the level of LOX-1 and CD36 on the surface of macrophages decreased, and the MCP-1, IL-6 and TNF- alpha in macrophages were decreased, and IL-10 increased. The NO generation and PGC-1 alpha expression of the human aortic skin cells were incubated with Ang II. The levels of cGMP and nitrite in endothelial cells increased after overexpression of PGC-1 a, suggesting that the inhibition of Ang II to reduce NO formation,.PGC-1 alpha, can block the dephosphorylation of eNOS, PI3K and Akt mediated by Ang II. Conclusion: high fat diet can lead to abnormal function of fat around blood vessels, and promote vasoconstriction and plaque formation. PRDM16 can induce the activation of brown fat in the fat around the abdominal aorta, inhibit the growth of the plaque and protect the vasoconstriction function of.3T3-L1 cells to differentiate into brown adipocytes after PRDM16 expression, and inhibit the inflammatory state of macrophages. Endothelial cells overexpress PGC-1 alpha by inhibiting the dephosphorylation level of eNOS through the PI3K-Akt pathway. It protects the function of endothelial function.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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