發(fā)布時(shí)間：2018-06-03 23:06

  本文選題：棕色脂肪組織 + 血管周?chē)窘M織��； 參考：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：背景:全球范圍內(nèi)以動(dòng)脈粥樣硬化為病理基礎(chǔ)的心腦血管疾病逐年增多,已經(jīng)成為危害人類健康的首位疾病。普遍認(rèn)為動(dòng)脈粥樣硬化病變始于內(nèi)膜損傷誘發(fā)的炎癥細(xì)胞浸潤(rùn)及脂質(zhì)沉積,整個(gè)過(guò)程是“由內(nèi)而外”的。但是,越來(lái)越多的證據(jù)顯示始于外膜的病變也可導(dǎo)致動(dòng)脈粥樣硬化斑塊的形成,其發(fā)生發(fā)展也可是“由外而內(nèi)”的。體內(nèi)大多數(shù)動(dòng)脈外層均有脂肪包裹,稱之為血管周?chē)�。既往認(rèn)為脂肪組織的主要功能是保溫及機(jī)械保護(hù),深入的研究發(fā)現(xiàn)脂肪組織是體內(nèi)重要的內(nèi)分泌器官,調(diào)節(jié)多種生理功能。血管周?chē)九c其他部位脂肪組織相似可以通過(guò)內(nèi)分泌及旁分泌眾多細(xì)胞因子或者脂肪因子直接調(diào)節(jié)血管功能。人體內(nèi)脂肪主要分為白色脂肪及棕色脂肪,活化棕色脂肪可改善代謝功能,降低血糖及血脂,抑制肥胖。近年來(lái)的研究已經(jīng)較為明確的闡明了棕色脂肪功能細(xì)胞活化及分化調(diào)節(jié)的相關(guān)機(jī)制,使得活化棕色脂肪功能治療疾病成為可能。 目的:通過(guò)β3受體激動(dòng)劑及轉(zhuǎn)基因方法(PRDM16)誘導(dǎo)血管周?chē)窘M織中棕色脂肪功能活化,觀察棕色脂肪功能活化對(duì)血管舒縮功能及動(dòng)脈粥樣硬化病變的影響。轉(zhuǎn)染PRDM16誘導(dǎo)脂肪前體細(xì)胞分化成為棕色脂肪細(xì),觀察對(duì)內(nèi)皮細(xì)胞及巨噬細(xì)胞的功能的影響。觀察棕色脂肪調(diào)控基因PGC-1α對(duì)內(nèi)皮功能的作用。方法:C57BL/6J小鼠隨機(jī)分成三組,分別予以正常飲食,高脂飲食以及高脂喂養(yǎng)+腹主動(dòng)脈周?chē)窘M織中CL-316243注射組。Apo E-/-小鼠隨機(jī)分成腹主動(dòng)脈周?chē)窘M織假手術(shù)處理(生理鹽水),空腺病毒載體轉(zhuǎn)染以及PRDM16腺病毒載體轉(zhuǎn)染。每周稱重,實(shí)驗(yàn)終點(diǎn)時(shí)留取血標(biāo)本測(cè)定血脂及血糖。HE染色觀察主動(dòng)脈斑塊及周?chē)窘M織。通過(guò)腹主動(dòng)脈環(huán)線性肌動(dòng)描記測(cè)定離體血管收縮及舒張功能。RT-PCR測(cè)定脂肪分泌因子如:瘦素、脂聯(lián)素、MCP-1、IL-8、TNF-α、IL-6以及IL-10。在3T3-L1細(xì)胞中過(guò)表達(dá)PRDM16誘導(dǎo)分化成為棕色脂肪細(xì)胞并與內(nèi)皮細(xì)胞及巨噬細(xì)胞共培養(yǎng)。測(cè)定內(nèi)皮細(xì)胞NO,eNOS,ET-1水平以及巨噬細(xì)胞表面LOX-1及CD36水平,同時(shí)測(cè)定巨噬細(xì)胞M1及M2極化狀態(tài)。人主動(dòng)脈內(nèi)皮細(xì)胞轉(zhuǎn)染PGC-1α后觀察血管緊張素Ⅱ處理對(duì)于NO生成的影響,以及對(duì)內(nèi)皮eNOS,PI3K以及Akt蛋白和mRNA水平的調(diào)控。結(jié)果:相對(duì)于正常飲食,高脂飲食顯著增加小鼠體重,提高血脂血糖及血壓水平,增加腹主動(dòng)脈周?chē)矩?fù)荷,促使瘦素及炎癥因子水平增高,脂聯(lián)素水平降低。CL-316243腹主動(dòng)脈周?chē)⑸湮茨茱@著改變小鼠代謝指標(biāo),也未能抑制脂肪組織炎癥狀態(tài)及改善內(nèi)分泌功能。但是PVAT可誘導(dǎo)脂肪細(xì)胞向棕色脂肪樣細(xì)胞分化,棕色脂肪基因UCP-1及線粒體水平增高。與PRDM16組相比,對(duì)照組及空病毒組腹主動(dòng)脈斑塊顯著增多。PRDM16組腹主動(dòng)脈PVAT中以棕色脂肪樣細(xì)胞為主,而其他兩組以白色脂肪細(xì)胞為主。PRDM16組PE或者KCl介導(dǎo)的腹主動(dòng)脈血管環(huán)收縮顯著減弱,而ACh介導(dǎo)的血管舒張?jiān)鰪?qiáng)。PRDM16轉(zhuǎn)染后顯著增高瘦素、脂聯(lián)素以及IL-8 mRNA水平,降低MCP-1,TNF-α,IL-6以及IL-10mRNA水平。PRDM16轉(zhuǎn)染3T3-L1細(xì)胞可誘導(dǎo)細(xì)胞向棕色脂肪細(xì)胞分化。將巨噬細(xì)胞與PRDM16誘導(dǎo)分化的棕色脂肪細(xì)胞共培養(yǎng),巨噬細(xì)胞表面LOX-1和CD36水平下降,此外巨噬細(xì)胞內(nèi)MCP-1,IL-6及TNF-α下降,而IL-10增高。將人主動(dòng)脈內(nèi)皮細(xì)胞與AngⅡ共孵育后NO生成及PGC-1α表達(dá)水平均下調(diào),過(guò)表達(dá)PGC-1α后內(nèi)皮細(xì)胞內(nèi)cGMP及亞硝酸鹽含量水平增高,提示對(duì)AngⅡ降低NO生成的抑制作用。PGC-1α可阻斷AngⅡ介導(dǎo)的eNOS,PI3K以及Akt的去磷酸化。結(jié)論:高脂飲食可導(dǎo)致血管周?chē)竟δ墚惓?并促進(jìn)血管收縮及斑塊形成。轉(zhuǎn)染PRDM16可誘導(dǎo)腹主動(dòng)脈周?chē)局凶厣竟δ芑罨?抑制斑塊生長(zhǎng)并保護(hù)血管舒縮功能。3T3-L1細(xì)胞表達(dá)PRDM16后可分化為棕色脂肪細(xì)胞,并抑制巨噬細(xì)胞的炎癥狀態(tài)。內(nèi)皮細(xì)胞過(guò)表達(dá)PGC-1α通過(guò)PI3K-Akt途徑抑制eNOS的去磷酸化水平,發(fā)揮其保護(hù)內(nèi)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R54

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2 胡明s

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