【摘要】：血管內(nèi)皮細(xì)胞是襯于血管內(nèi)表面的單層扁平上皮細(xì)胞,是血管的第一道屏障,在維持血管穩(wěn)態(tài)中發(fā)揮重要作用。內(nèi)皮細(xì)胞功能失常是心血管疾病早期的代表性特征,內(nèi)皮細(xì)胞衰老被認(rèn)為是導(dǎo)致其功能失常的重要因素。在人的動(dòng)脈粥樣硬化斑塊組織、高脂血癥和糖尿病性血管中均檢測(cè)到了衰老的血管內(nèi)皮細(xì)胞,說(shuō)明內(nèi)皮細(xì)胞的衰老可能與血管疾病的病理生理過(guò)程關(guān)系緊密。哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是一種絲氨酸/蘇氨酸蛋白激酶,在細(xì)胞生長(zhǎng)、分化、增殖、遷移和存活中扮演重要角色。mTOR有兩種不同的多蛋白復(fù)合體——mTORC1和mTORC2。RICTOR(Rapamycin-insensitive companion of mTOR)是mTORC2復(fù)合物的重要組成部分,可以磷酸化并激活A(yù)KT。AKT能直接磷酸化多種轉(zhuǎn)錄因子,通過(guò)調(diào)控這些轉(zhuǎn)錄因子,可以抑制凋亡基因的表達(dá)和(或)增強(qiáng)抗凋亡基因的表達(dá),從而促進(jìn)細(xì)胞的存活。然而,RICTOR能否在抑制內(nèi)皮細(xì)胞衰老的過(guò)程中發(fā)揮作用則鮮有報(bào)道。為了探討RICTOR在血管內(nèi)皮衰老中的作用,我們建立了體外人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cell,HUVEC)的自然衰老細(xì)胞模型、高糖(high glucose,HG)和ox-LDL(oxidized low density lipoprotein,ox-LDL)刺激的細(xì)胞模型以及體內(nèi)C57野生型小鼠主動(dòng)脈的自然衰老模型。我們研究發(fā)現(xiàn)在上述體外細(xì)胞模型中RICTOR表達(dá)下降(P0.001,0.05,0.01),這提示RICTOR可能在調(diào)節(jié)血管內(nèi)皮衰老中發(fā)揮重要作用。隨后我們構(gòu)建了RICTOR過(guò)表達(dá)腺病毒載體,對(duì)上述三種細(xì)胞模型中RICTOR的表達(dá)進(jìn)行糾正,結(jié)果顯示RICTOR、AKT及其下游TERT、eNOS等蛋白表達(dá)均有不同程度改變。我們又糾正自然衰老小鼠血管內(nèi)皮RICTOR的表達(dá),發(fā)現(xiàn)血管內(nèi)皮依賴的舒張功能得到改善(P0.05)。這些結(jié)果證明內(nèi)皮細(xì)胞中RICTOR的特異性過(guò)表達(dá)對(duì)衰老有抑制作用。近年來(lái),越來(lái)越多的研究表明microRNA(miRNA)在細(xì)胞衰老中發(fā)揮重要作用。通過(guò)篩選,我們發(fā)現(xiàn)在衰老的血管內(nèi)皮細(xì)胞中,miRNA-152的表達(dá)異常升高,提示其可能在血管內(nèi)皮衰老發(fā)展過(guò)程中發(fā)揮促進(jìn)作用。因此,我們猜測(cè)內(nèi)皮細(xì)胞中RICTOR的特異性過(guò)表達(dá)對(duì)衰老的抑制作用可能是miRNA-152的下調(diào)引起的。為了驗(yàn)證此假設(shè),我們研究了 miRNA-152對(duì)RICTOR的調(diào)節(jié)作用。我們發(fā)現(xiàn)miRNA-152對(duì)RICTOR有負(fù)性調(diào)節(jié)作用,進(jìn)一步雙熒光素酶報(bào)告基因分析發(fā)現(xiàn),RICTOR是miRNA-152的靶基因。本研究中我們首次發(fā)現(xiàn)在內(nèi)皮細(xì)胞中過(guò)表達(dá)RICTOR能夠抑制內(nèi)皮細(xì)胞老化并改善因衰老導(dǎo)致的內(nèi)皮功能失常。同時(shí),我們首次證明了血管內(nèi)皮細(xì)胞中RICTOR的表達(dá)可以由miRNA-152調(diào)控。
[Abstract]:Vascular endothelial cells are monolayer flat epithelial cells lined on the inner surface of blood vessels. They are the first barrier of blood vessels and play an important role in maintaining vascular homeostasis. Endothelial cell dysfunction is a representative feature of cardiovascular disease in the early stage, and endothelial cell aging is considered to be an important factor leading to its dysfunction. Aging vascular endothelial cells were detected in human atherosclerotic plaques, hyperlipidemia and diabetic vessels, indicating that the aging of endothelial cells may be closely related to the pathophysiological process of vascular diseases. Mammal rapamycin target protein (mammalian target of rapamycin,mTOR is a serine / threonine protein kinase, which plays an important role in cell growth, differentiation, proliferation, migration and survival. MTOR has two different polyprotein complexes, mTORC1 and mTORC2.RICTOR (Rapamycin-insensitive companion of mTOR) is an important part of mTORC2 complex, which can phosphorylation and activate AKT.AKT and directly phosphorylation a variety of transcription factors. By regulating these transcription factors, the expression of apoptotic genes can be inhibited and / or the expression of anti-apoptotic genes can be enhanced, thus promoting the survival of cells. However, it is rarely reported whether RICTOR can play a role in inhibiting the senescence of endothelial cells. In order to investigate the role of RICTOR in vascular endothelial aging, we established the natural aging cell model of human umbilical vein endothelial cell (human umbilical vein endothelial cell,HUVEC) in vitro, the cell model stimulated by high glucose (high glucose,HG) and ox-LDL (oxidized low density lipoprotein,ox-LDL, and the natural aging model of the aortic of C57 wild type mice in vivo. Our study found that the expression of RICTOR decreased in the above cell model in vitro (P0.0010.050.001), which suggests that RICTOR may play an important role in the regulation of vascular endothelial senescence. Then we constructed RICTOR overexpression adenoviral vector and corrected the expression of RICTOR in the above three cell models. The results showed that the expression of RICTOR,AKT and its downstream TERT,eNOS proteins changed in varying degrees. We also corrected the expression of vascular endothelial RICTOR in natural aging mice, and found that the vasodilation function was improved (P 0.05). These results suggest that the specific overexpression of RICTOR in endothelial cells can inhibit aging. In recent years, more and more studies have shown that microRNA (miRNA) plays an important role in cell senescence. Through screening, we found that the expression of miRNA-152 increased abnormally in aging vascular endothelial cells, suggesting that it may play a role in promoting the development of vascular endothelial aging. Therefore, we speculate that the inhibitory effect of RICTOR specific overexpression on aging in endothelial cells may be caused by the down-regulation of miRNA-152. In order to verify this hypothesis, we studied the regulatory effect of miRNA-152 on RICTOR. We found that miRNA-152 had a negative regulatory effect on RICTOR. Further analysis of double luciferase reporter gene showed that RICTOR was the target gene of miRNA-152. In this study, we found for the first time that overexpression of RICTOR in endothelial cells can inhibit the aging of endothelial cells and improve endothelial dysfunction caused by aging. At the same time, we proved for the first time that the expression of RICTOR in vascular endothelial cells could be regulated by miRNA-152.
【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)】：R54

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本文編號(hào)：2500661