RICTOR对人血管内皮细胞衰老的调控作用
发布时间：2019-06-16 16:13
【摘要】：血管内皮细胞是衬于血管内表面的单层扁平上皮细胞,是血管的第一道屏障,在维持血管稳态中发挥重要作用。内皮细胞功能失常是心血管疾病早期的代表性特征,内皮细胞衰老被认为是导致其功能失常的重要因素。在人的动脉粥样硬化斑块组织、高脂血症和糖尿病性血管中均检测到了衰老的血管内皮细胞,说明内皮细胞的衰老可能与血管疾病的病理生理过程关系紧密。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是一种丝氨酸/苏氨酸蛋白激酶,在细胞生长、分化、增殖、迁移和存活中扮演重要角色。mTOR有两种不同的多蛋白复合体——mTORC1和mTORC2。RICTOR(Rapamycin-insensitive companion of mTOR)是mTORC2复合物的重要组成部分,可以磷酸化并激活AKT。AKT能直接磷酸化多种转录因子,通过调控这些转录因子,可以抑制凋亡基因的表达和(或)增强抗凋亡基因的表达,从而促进细胞的存活。然而,RICTOR能否在抑制内皮细胞衰老的过程中发挥作用则鲜有报道。为了探讨RICTOR在血管内皮衰老中的作用,我们建立了体外人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)的自然衰老细胞模型、高糖(high glucose,HG)和ox-LDL(oxidized low density lipoprotein,ox-LDL)刺激的细胞模型以及体内C57野生型小鼠主动脉的自然衰老模型。我们研究发现在上述体外细胞模型中RICTOR表达下降(P0.001,0.05,0.01),这提示RICTOR可能在调节血管内皮衰老中发挥重要作用。随后我们构建了RICTOR过表达腺病毒载体,对上述三种细胞模型中RICTOR的表达进行纠正,结果显示RICTOR、AKT及其下游TERT、eNOS等蛋白表达均有不同程度改变。我们又纠正自然衰老小鼠血管内皮RICTOR的表达,发现血管内皮依赖的舒张功能得到改善(P0.05)。这些结果证明内皮细胞中RICTOR的特异性过表达对衰老有抑制作用。近年来,越来越多的研究表明microRNA(miRNA)在细胞衰老中发挥重要作用。通过筛选,我们发现在衰老的血管内皮细胞中,miRNA-152的表达异常升高,提示其可能在血管内皮衰老发展过程中发挥促进作用。因此,我们猜测内皮细胞中RICTOR的特异性过表达对衰老的抑制作用可能是miRNA-152的下调引起的。为了验证此假设,我们研究了 miRNA-152对RICTOR的调节作用。我们发现miRNA-152对RICTOR有负性调节作用,进一步双荧光素酶报告基因分析发现,RICTOR是miRNA-152的靶基因。本研究中我们首次发现在内皮细胞中过表达RICTOR能够抑制内皮细胞老化并改善因衰老导致的内皮功能失常。同时,我们首次证明了血管内皮细胞中RICTOR的表达可以由miRNA-152调控。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
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