Hes-1介導VEGF促進OPN表達在動脈粥樣硬化發(fā)生發(fā)展過程中的作用及分子機制研究
發(fā)布時間：2018-07-23 18:09
【摘要】：研究背景和目的:動脈粥樣硬化是導致心血管疾病的最關鍵致病因素。新生血管形成是粥樣硬化斑塊特征之一,它可以引發(fā)易損斑塊的發(fā)展,增加斑塊破裂的風險。但是,血管新生發(fā)生及發(fā)展的調(diào)控機制直至今日未明確。DNA結(jié)合蛋白(hairy enhancer of split,Hes-1)是一類DNA結(jié)合蛋白,因其結(jié)構(gòu)中含有α螺旋-環(huán)-螺旋(α HLH)而極具特征性。這一物質(zhì)可結(jié)合在特定分化效應基因的啟動子上,從而發(fā)揮介導細胞分化的抑制信號,并參與血管細胞發(fā)育、分化以及及新生血管形成等重要生理過程。Hes-1的調(diào)節(jié)轉(zhuǎn)錄在心血管以及平滑肌細胞的分化、血管的形成、動靜脈血管細胞的凋亡以及血管成形的過程中起到至關重要的作用。目前的一些研究已表明,Hes-1涉及了調(diào)節(jié)內(nèi)皮細胞(EC)的增殖、分化和凋亡的過程。但是,直至今日,關于HES-1是否參與到不穩(wěn)定斑塊中血管生成的過程中仍不清楚。骨橋蛋白(osteopontin,OPN)是一種分泌型糖基化磷蛋白,存在于細胞外基質(zhì)中,其作用是參與介導細胞的黏附、增殖以及遷移,對于調(diào)節(jié)血管重構(gòu)起重要作用。初步研究表明,OPN不僅促進動脈粥樣硬化,還促進了新生血管的生成。在內(nèi)皮發(fā)生損傷后,平滑肌細胞出現(xiàn)一系列去分化、遷移、增殖的過程,在這些過程中OPN通過介導細胞與細胞、細胞與基質(zhì)的相互作用進而發(fā)揮了促進血管重塑的作用。雖然OPN調(diào)控機制在血管生成過程中仍不清楚,有研究顯示Hes-1過表達抑制成骨細胞OPN轉(zhuǎn)錄,這表明Hes-1可能是OPN潛在調(diào)節(jié)因子。在動脈粥樣硬化斑塊中,內(nèi)皮細胞以及炎性細胞可以分泌產(chǎn)生血管內(nèi)皮生長因子(VEGF),其可作為始動因子參與是斑塊以及血管生成,與此同時Hes-1也參與了 VEGF的信號轉(zhuǎn)導。除了 hes-1,越來越多的研究發(fā)現(xiàn)VEGF誘導OPN的表達。然而,Hes-1是否通過調(diào)節(jié)OPN進而影響VEGF誘導的動脈粥樣硬化斑塊中新生血管生成尚未闡明。本課題研究擬通過分別檢測Hes-1和OPN在動脈粥樣硬化組織中的表達情況,進而分析這兩種物質(zhì)的表達與相關病理參數(shù)之間的內(nèi)在聯(lián)系,與此同時通過進行體外模型的實驗,研究并揭示Hes-1和OPN在AS疾病的發(fā)生、發(fā)展過程中的所發(fā)揮的作用和分子學相關機制。方法1.通過使用組織基因芯片來分析Hes-1和OPN為粥樣硬化動脈組織對比正常動脈組織標本中的差異性表達基因;2.熒光定量qPCR、免疫組織化學(IHC)和免疫熒光(immunofluorescence)檢測粥樣硬化動脈組織與正常動脈組織樣本中Hes-1和OPN的表達,并分析其的表達量、定位;3.通過體外VEGF刺激的方法,利用免疫印跡試驗(Westernblot)檢測Hes-1和OPN表達情況;4.構(gòu)筑建造Hes-1過表達型的載體,并使用合成siRNA對Hes-1的表達進行干擾,對臍靜脈內(nèi)皮細胞進行瞬時轉(zhuǎn)染,利用western blot方法檢測Hes-1對OPN的調(diào)控作用;5.構(gòu)建OPN過表達載體,合成siRNA干擾OPN的表達,通過基質(zhì)膠成管實驗檢測VEGF-Hes-1-OPN對血管生成的影響。結(jié)果基因芯片、RT-PCR以及IHC檢測的結(jié)果顯示:在動脈粥樣硬化斑塊的樣本中,Hes-1的表達水平顯著性降低,但是OPN的表達卻明顯增強。同時,免疫熒光結(jié)果顯示,Hes-1和OPN均在斑塊中新生血管的內(nèi)皮中表達。體外細胞實驗表明,在VEGF的刺激下,臍靜脈內(nèi)皮細胞中Hes-1呈時間及劑量依賴性降低,OPN呈時間及劑量依賴性升高。將Hes-1基因表達進行敲低處理后,可以增強OPN的表達以及VEGF誘導性下的血管生成。與此相反的是,如Hes-1基因過表達則會對OPN的表達起抑制作用并對VEGF誘導的血管生成起負性調(diào)節(jié)作用。結(jié)論動脈粥樣斑塊組織中Hes-1抑制了 OPN的表達,并在此基礎上抑制VEGF誘導的血管生成的發(fā)生及發(fā)展。因此,恢復Hes-1的表達、抑制OPN基因可作為預防不穩(wěn)定斑塊形成的重要舉措。
[Abstract]:Background and objective: atherosclerosis is the most important cause of cardiovascular disease. Neovascularization is one of the characteristics of atherosclerotic plaque. It can cause the development of vulnerable plaque and increase the risk of plaque rupture. However, the regulation mechanism of angiogenesis and development of angiogenesis is not clear to.DNA binding protein until today (hairy Enhancer of split, Hes-1) is a class of DNA binding proteins which are characterized by its structure containing alpha spiral loop helix (alpha HLH). This substance can be combined with the promoter of a specific differentiation effect gene to play an inhibitory signal to mediate cell differentiation, and to participate in the development of vascular cells, differentiation, and the formation of neovascularization. Regulatory transcription of.Hes-1 is essential for the differentiation of cardiovascular and smooth muscle cells, the formation of blood vessels, the apoptosis of arteriovenous cells and the process of angioplasty. Some studies have shown that Hes-1 involves the process of regulating the proliferation, differentiation and apoptosis of endothelial cells (EC), but to this day, It is not clear whether HES-1 is involved in angiogenesis in unstable plaques. Osteopontin (OPN) is a kind of secreted glycosylated phosphoprotein, which exists in the extracellular matrix. Its role is to mediate cell adhesion, proliferation and migration, and plays an important role in regulating vascular remodeling. Preliminary studies have shown that OPN does not It only promotes atherosclerosis and promotes the formation of new blood vessels. After endothelial damage, the smooth muscle cells have a series of processes of dedifferentiation, migration and proliferation. In these processes, OPN plays a role in promoting vascular remodeling by mediating the interaction between cells and cells, cells and the matrix. Although the mechanism of OPN regulation is in blood Guan Shengcheng's process is still unclear. Studies have shown that overexpression of Hes-1 inhibits osteoblast OPN transcription, which suggests that Hes-1 may be a potential regulator of OPN. In atherosclerotic plaques, endothelial cells and inflammatory cells can secrete the production of vascular endothelial growth factor (VEGF), which can be involved in plaque and angiogenesis as a initiating factor. At the same time, Hes-1 also participates in the signal transduction of VEGF. In addition to HES-1, more and more studies have found that VEGF induces the expression of OPN. However, Hes-1 has not been elucidated by regulating OPN and thus affecting the angiogenesis in VEGF induced atherosclerotic plaques. This study is to detect Hes-1 and OPN in atherosclerotic atherosclerosis by detecting Hes-1 and OPN respectively. And then analyze the expression in the tissue, and then analyze the internal relationship between the expression of the two substances and the related pathological parameters. At the same time, through the experiment of the model in vitro, the role of Hes-1 and OPN in the development of AS disease, the role of the molecular mechanism in the development process and the molecular mechanism are revealed. Method 1. through the use of the tissue gene chip To analyze the differential expression genes of Hes-1 and OPN in atherosclerotic arterial tissue compared with normal arterial tissue, 2. fluorescence quantitative qPCR, immunohistochemistry (IHC) and immunofluorescence (immunofluorescence) were used to detect the expression of Hes-1 and OPN in atherosclerotic arterial tissue and normal arterial tissue samples, and to analyze the expression and location of 3.. The expression of Hes-1 and OPN was detected by the method of VEGF stimulation in vitro, and the expression of Hes-1 and OPN was detected by Western blot test (Westernblot); 4. the Hes-1 overexpression vector was constructed and siRNA was used to interfere with the expression of Hes-1, and the umbilical vein endothelial cells were transiently transfected, and Western blot method was used to detect Hes-1 to OPN, and 5. to construct O. PN overexpressed the expression vector, synthesized the expression of siRNA interfering OPN, and detected the effect of VEGF-Hes-1-OPN on angiogenesis. Results the results of gene chip, RT-PCR and IHC showed that the expression level of Hes-1 was significantly decreased in the samples of atherosclerotic plaque, but the expression of OPN was obviously enhanced. Meanwhile, the immune system was immune. The fluorescence results showed that both Hes-1 and OPN were expressed in the endothelial cells of the neovascularization in the plaque. In vitro cell experiments showed that under the stimulation of VEGF, the Hes-1 in the umbilical vein endothelial cells decreased in time and dose dependence, and the OPN was increased in time and dose dependence. The expression of Hes-1 gene and the VEGF could enhance the expression of OPN and VEGF. In contrast, the overexpression of Hes-1 gene can inhibit the expression of OPN and negatively regulate the angiogenesis induced by VEGF. Conclusion Hes-1 inhibits the expression of OPN in the atherosclerotic plaque tissue and inhibits the occurrence and development of angiogenesis induced by VEGF. The expression of compound Hes-1 and inhibition of OPN gene can be used as an important measure to prevent the formation of unstable plaque.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R543.5
本文編號：2140211
[Abstract]:Background and objective: atherosclerosis is the most important cause of cardiovascular disease. Neovascularization is one of the characteristics of atherosclerotic plaque. It can cause the development of vulnerable plaque and increase the risk of plaque rupture. However, the regulation mechanism of angiogenesis and development of angiogenesis is not clear to.DNA binding protein until today (hairy Enhancer of split, Hes-1) is a class of DNA binding proteins which are characterized by its structure containing alpha spiral loop helix (alpha HLH). This substance can be combined with the promoter of a specific differentiation effect gene to play an inhibitory signal to mediate cell differentiation, and to participate in the development of vascular cells, differentiation, and the formation of neovascularization. Regulatory transcription of.Hes-1 is essential for the differentiation of cardiovascular and smooth muscle cells, the formation of blood vessels, the apoptosis of arteriovenous cells and the process of angioplasty. Some studies have shown that Hes-1 involves the process of regulating the proliferation, differentiation and apoptosis of endothelial cells (EC), but to this day, It is not clear whether HES-1 is involved in angiogenesis in unstable plaques. Osteopontin (OPN) is a kind of secreted glycosylated phosphoprotein, which exists in the extracellular matrix. Its role is to mediate cell adhesion, proliferation and migration, and plays an important role in regulating vascular remodeling. Preliminary studies have shown that OPN does not It only promotes atherosclerosis and promotes the formation of new blood vessels. After endothelial damage, the smooth muscle cells have a series of processes of dedifferentiation, migration and proliferation. In these processes, OPN plays a role in promoting vascular remodeling by mediating the interaction between cells and cells, cells and the matrix. Although the mechanism of OPN regulation is in blood Guan Shengcheng's process is still unclear. Studies have shown that overexpression of Hes-1 inhibits osteoblast OPN transcription, which suggests that Hes-1 may be a potential regulator of OPN. In atherosclerotic plaques, endothelial cells and inflammatory cells can secrete the production of vascular endothelial growth factor (VEGF), which can be involved in plaque and angiogenesis as a initiating factor. At the same time, Hes-1 also participates in the signal transduction of VEGF. In addition to HES-1, more and more studies have found that VEGF induces the expression of OPN. However, Hes-1 has not been elucidated by regulating OPN and thus affecting the angiogenesis in VEGF induced atherosclerotic plaques. This study is to detect Hes-1 and OPN in atherosclerotic atherosclerosis by detecting Hes-1 and OPN respectively. And then analyze the expression in the tissue, and then analyze the internal relationship between the expression of the two substances and the related pathological parameters. At the same time, through the experiment of the model in vitro, the role of Hes-1 and OPN in the development of AS disease, the role of the molecular mechanism in the development process and the molecular mechanism are revealed. Method 1. through the use of the tissue gene chip To analyze the differential expression genes of Hes-1 and OPN in atherosclerotic arterial tissue compared with normal arterial tissue, 2. fluorescence quantitative qPCR, immunohistochemistry (IHC) and immunofluorescence (immunofluorescence) were used to detect the expression of Hes-1 and OPN in atherosclerotic arterial tissue and normal arterial tissue samples, and to analyze the expression and location of 3.. The expression of Hes-1 and OPN was detected by the method of VEGF stimulation in vitro, and the expression of Hes-1 and OPN was detected by Western blot test (Westernblot); 4. the Hes-1 overexpression vector was constructed and siRNA was used to interfere with the expression of Hes-1, and the umbilical vein endothelial cells were transiently transfected, and Western blot method was used to detect Hes-1 to OPN, and 5. to construct O. PN overexpressed the expression vector, synthesized the expression of siRNA interfering OPN, and detected the effect of VEGF-Hes-1-OPN on angiogenesis. Results the results of gene chip, RT-PCR and IHC showed that the expression level of Hes-1 was significantly decreased in the samples of atherosclerotic plaque, but the expression of OPN was obviously enhanced. Meanwhile, the immune system was immune. The fluorescence results showed that both Hes-1 and OPN were expressed in the endothelial cells of the neovascularization in the plaque. In vitro cell experiments showed that under the stimulation of VEGF, the Hes-1 in the umbilical vein endothelial cells decreased in time and dose dependence, and the OPN was increased in time and dose dependence. The expression of Hes-1 gene and the VEGF could enhance the expression of OPN and VEGF. In contrast, the overexpression of Hes-1 gene can inhibit the expression of OPN and negatively regulate the angiogenesis induced by VEGF. Conclusion Hes-1 inhibits the expression of OPN in the atherosclerotic plaque tissue and inhibits the occurrence and development of angiogenesis induced by VEGF. The expression of compound Hes-1 and inhibition of OPN gene can be used as an important measure to prevent the formation of unstable plaque.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R543.5
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