RICTOR對人血管內(nèi)皮細胞衰老的調(diào)控作用
發(fā)布時間：2019-06-16 16:13
【摘要】：血管內(nèi)皮細胞是襯于血管內(nèi)表面的單層扁平上皮細胞,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)皮細胞功能失常是心血管疾病早期的代表性特征,內(nèi)皮細胞衰老被認為是導致其功能失常的重要因素。在人的動脈粥樣硬化斑塊組織、高脂血癥和糖尿病性血管中均檢測到了衰老的血管內(nèi)皮細胞,說明內(nèi)皮細胞的衰老可能與血管疾病的病理生理過程關(guān)系緊密。哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是一種絲氨酸/蘇氨酸蛋白激酶,在細胞生長、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合體——mTORC1和mTORC2。RICTOR(Rapamycin-insensitive companion of mTOR)是mTORC2復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,通過調(diào)控這些轉(zhuǎn)錄因子,可以抑制凋亡基因的表達和(或)增強抗凋亡基因的表達,從而促進細胞的存活。然而,RICTOR能否在抑制內(nèi)皮細胞衰老的過程中發(fā)揮作用則鮮有報道。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)皮細胞(human umbilical vein endothelial cell,HUVEC)的自然衰老細胞模型、高糖(high glucose,HG)和ox-LDL(oxidized low density lipoprotein,ox-LDL)刺激的細胞模型以及體內(nèi)C57野生型小鼠主動脈的自然衰老模型。我們研究發(fā)現(xiàn)在上述體外細胞模型中RICTOR表達下降(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過表達腺病毒載體,對上述三種細胞模型中RICTOR的表達進行糾正,結(jié)果顯示RICTOR、AKT及其下游TERT、eNOS等蛋白表達均有不同程度改變。我們又糾正自然衰老小鼠血管內(nèi)皮RICTOR的表達,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)皮細胞中RICTOR的特異性過表達對衰老有抑制作用。近年來,越來越多的研究表明microRNA(miRNA)在細胞衰老中發(fā)揮重要作用。通過篩選,我們發(fā)現(xiàn)在衰老的血管內(nèi)皮細胞中,miRNA-152的表達異常升高,提示其可能在血管內(nèi)皮衰老發(fā)展過程中發(fā)揮促進作用。因此,我們猜測內(nèi)皮細胞中RICTOR的特異性過表達對衰老的抑制作用可能是miRNA-152的下調(diào)引起的。為了驗證此假設(shè),我們研究了 miRNA-152對RICTOR的調(diào)節(jié)作用。我們發(fā)現(xiàn)miRNA-152對RICTOR有負性調(diào)節(jié)作用,進一步雙熒光素酶報告基因分析發(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)皮細胞中過表達RICTOR能夠抑制內(nèi)皮細胞老化并改善因衰老導致的內(nèi)皮功能失常。同時,我們首次證明了血管內(nèi)皮細胞中RICTOR的表達可以由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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
本文編號：2500661
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
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