SIRT1在血管平滑肌細(xì)胞增殖和新生內(nèi)膜形成中的作用及分子機(jī)制研究
發(fā)布時間：2018-12-19 08:02
【摘要】：新生內(nèi)膜形成與血管重塑密切相關(guān),主要包括血管平滑肌細(xì)胞的增殖、遷移、凋亡以及基質(zhì)成分合成、降解及重新排列等過程。過度的血管平滑肌細(xì)胞增殖是動脈粥樣硬化、肺動脈高壓、系統(tǒng)性高血壓、腹主動脈瘤及經(jīng)皮腔內(nèi)血管成形術(shù)后再狹窄等心血管疾病的重要病理基礎(chǔ)。細(xì)胞增殖主要通過以下三種蛋白進(jìn)行調(diào)控：周期蛋白依賴性蛋白激酶(Cyclin-dependent kinase, CDK),周期蛋白(Cyclin),周期蛋白依賴性蛋白激酶抑制因子(Cyclin-dependent kinase inhibitor, CKI)。其中CyclinDl在細(xì)胞周期中最早被合成,于G1中期到達(dá)高峰,并與CDK4/CDK6形成復(fù)合物,對決定G1/S期轉(zhuǎn)換的限制點(diǎn)進(jìn)行調(diào)控。CyclinDl主要有轉(zhuǎn)錄水平及泛素化降解途徑的調(diào)控,在生長因子刺激下,多種轉(zhuǎn)錄因子如：AP-1 (activator protein-1), NF-κB (nuclear factor-κB),SP1等激活CyclinDl,在轉(zhuǎn)錄水平調(diào)控CyclinDl的表達(dá),其中AP-1對其的調(diào)控研究最多。 人類SIRT1 (SIRTuin 1)是NAD+依賴的Ⅲ類組蛋白去乙酰化酶,在各物種之間呈現(xiàn)高度的保守性,在7個SIRT成員中與酵母Sir2 (silence information regulator2)的同源性最高。近年來的研究發(fā)現(xiàn),SIRT1能夠去乙�；M蛋白(H3,H4)和多種轉(zhuǎn)錄因子和轉(zhuǎn)錄輔助因子,包括MyoD、p300、PGC-1α、PPARγ、NF-κB、Ku70、p53、FOXO、E2F1等,在胚胎發(fā)育、組織分化、代謝調(diào)節(jié)、細(xì)胞凋亡、DNA損傷修復(fù)和抵抗應(yīng)激方面發(fā)揮重要的作用。在多種細(xì)胞中的研究發(fā)現(xiàn),SIRT1還能夠直接調(diào)節(jié)細(xì)胞周期和細(xì)胞增殖。因此我們設(shè)想：在平滑肌細(xì)胞中過表達(dá)SIRT1能夠抑制細(xì)胞增殖,從而對抗血管增殖性疾病的增殖。 本研究首先應(yīng)用RT-PCR和Western blot檢測了SIRT1在血清處理的血管平滑肌細(xì)胞和結(jié)扎后的頸總動脈中的表達(dá)變化。隨后我們應(yīng)用RT-PCR、Southern blot、Western blot、免疫組化等方法對平滑肌特異性SIRT1轉(zhuǎn)基因鼠進(jìn)行鑒定,應(yīng)用小鼠頸總動脈結(jié)扎模型,采用HE染色、免疫組化、Masson染色、TUNEL染色等方法觀察轉(zhuǎn)基因鼠新生內(nèi)膜形成,血管平滑肌細(xì)胞增殖,細(xì)胞外基質(zhì)(ECM)及動脈中細(xì)胞凋亡。進(jìn)一步通過H3-TdR、流式細(xì)胞術(shù)檢測SIRT1在體外對細(xì)胞增殖和細(xì)胞周期的影響。隨后,通過Western blot、RT-PCR觀察在基礎(chǔ)水平和血清誘導(dǎo)下,過表達(dá)和干擾SIRT1對細(xì)胞周期蛋白表達(dá)水平的影響；用熒光素酶報告系統(tǒng)檢測對AP-1下游基因CyclinD1活性的影響；ChIP檢測SIRT1以及AP-1兩個亞基c-Fos/c-Jun在CyclinD1啟動子上的募集。 本研究發(fā)現(xiàn)血清處理的血管平滑肌細(xì)胞中SIRT1表達(dá)先升高,最后表達(dá)顯著降低,小鼠頸總動脈結(jié)扎模型中SIRT1表達(dá)隨新生內(nèi)膜形成短暫的升高后,顯著降低。實驗表明SIRT1轉(zhuǎn)基因鼠可以抑制由頸總動脈結(jié)扎所引起的新生內(nèi)膜形成,并且可以減少細(xì)胞外基質(zhì),但血管平滑肌細(xì)胞凋亡沒有顯著變化。腺病毒介導(dǎo)的SIRT1過表達(dá)可以抑制基礎(chǔ)水平和血清誘導(dǎo)的原代血管平滑肌細(xì)胞增殖,并使其血清誘導(dǎo)的原代血管平滑肌細(xì)胞阻滯在G1期。SIRT1在體內(nèi)外抑制G1期開關(guān)分子CyclinD1的表達(dá),進(jìn)一步研究表明SIRT1在血管平滑肌細(xì)胞中結(jié)合c-Fos/c-Jun并抑制其在CyclinD1啟動子上的募集,這可能是導(dǎo)致SIRT1抑制CyclinD1表達(dá)變化的分子基礎(chǔ)。 我們的研究發(fā)現(xiàn)小鼠頸總動脈結(jié)扎模型中SIRT1表達(dá)隨新生內(nèi)膜形成降低；SIRT1在體內(nèi)也可以降低CyclinD1的表達(dá),并可能由此抑制新生內(nèi)膜形成。在血管平滑肌細(xì)胞中,SIRT1通過抑制c-Fos/c-Jun在CyclinD1啟動子上的募集,從而降低CyclinD1的表達(dá),使血管平滑肌細(xì)胞阻滯在G1期,并進(jìn)而抑制血管平滑肌細(xì)胞的增殖。因此,SIRT1是調(diào)控平滑肌細(xì)胞增殖的重要基因,提高SIRT1的表達(dá)可以作為抑制血管平滑肌細(xì)胞增殖和新生內(nèi)膜形成,影響血管重塑過程的一個新手段。
[Abstract]:Neointimal formation is closely related to vascular remodeling, mainly including the proliferation, migration, apoptosis and the synthesis, degradation and rearrangement of vascular smooth muscle cells. Hypervascular smooth muscle cell proliferation is an important pathological basis for atherosclerosis, pulmonary hypertension, systemic hypertension, abdominal aortic aneurysm and restenosis after percutaneous transluminal angioplasty. Cell proliferation is mainly regulated by the following three proteins: Cyclin-dependent kinase (CDK), Cyclin (Cyclin), and Cyclin-dependent kinase inhibitor (CKI). CyclinDl was first synthesized in the cell cycle and reached the peak at the middle of G1, and the complex was formed with CDK4/ CDK6 to control the limit of the G1/ S phase transition. CyclinDl mainly has the regulation of transcription level and the degradation pathway of ubiquitination. Under the stimulation of growth factor, a variety of transcription factors such as AP-1 (activator protein-1), NF-B (nar factor-B), SP1 and the like activate Cyclin Dl, and the expression of Cyclin Dl is regulated at the transcription level, wherein the AP-1 has the most control and control. human SIRT1 (SIRTuin 1) is an NAD +-dependent type III histone de-ethylation enzyme, a high degree of conservation is present between species, and the homology with yeast Sir2 (sience information regulator2) in 7 SIRT members is most High. In recent years, it has been found that SIRT1 is capable of deethanizing histone (H3, H4) and various transcription factors and transcription factors, including MyoD, p300, PGC-1, PPAR, NF-B, Ku70, p53, FOXO, E2F1, etc., in embryonic development, tissue differentiation, metabolism regulation, and cell death. It plays an important role in the repair of death, DNA damage and the resistance to stress. It is found that SIRT1 can also directly regulate cell cycle and cell proliferation in a variety of cells. It is therefore envisaged that overexpression of SIRT1 in smooth muscle cells can inhibit cell proliferation, thereby increasing the proliferation of vascular proliferative diseases. In this study, RT-PCR and Western blot were used to detect the presence of SIRT1 in serum-treated vascular smooth muscle cells and the common carotid artery after ligation. The specific SIRT1 transgenic mice were identified by RT-PCR, Southern blot, Western blot and immunohistochemistry. membrane formation, vascular smooth muscle cell proliferation, extracellular matrix (ECM) and thin artery Cell apoptosis. The proliferation and cell cycle of SIRT1 in vitro were detected by flow cytometry with H3-TdR. The effect of SIRT1 on the level of cell cycle protein expression was observed by Western blot and RT-PCR, and the activity of Cyclin D1 in the downstream gene of AP-1 was detected by luciferase reporter system. Effect of two subunits, c-Fos/ c-Jun, on the Cyclin D1 promoter In this study, the expression of SIRT1 in the vascular smooth muscle cells treated with serum was increased, the final expression was significantly decreased, and the expression of SIRT1 in the model of the common carotid artery of the mouse increased with the formation of the neointima. The results show that the SIRT1 transgenic mice can inhibit the formation of neointima caused by the ligature of the common carotid artery and reduce the extracellular matrix, but the apoptosis of the vascular smooth muscle cells There is a significant change. The overexpression of SIRT1 mediated by adenovirus can inhibit the basal level and the proliferation of primary vascular smooth muscle cells induced by the serum, and make the serum-induced primary vascular smooth muscle cell resistance SIRT1 inhibits the expression of CyclinD1 in the G1 phase of the vascular smooth muscle cells, and further studies indicate that SIRT1 binds to c-Fos/ c-Jun in the vascular smooth muscle cells and inhibits its recruitment on the Cyclin D1 promoter, which may be the result of the inhibition of the expression of CyclinD1 by SIRT1 The molecular basis of the molecular basis. Our study found that the expression of SIRT1 in the rat carotid artery ligation model decreased with the formation of neointima, and the expression of Cyclin D1 could also be reduced in SIRT1 in vivo and may be suppressed. In vascular smooth muscle cells, SIRT1 reduces the expression of CyclinD1 by inhibiting the recruitment of c-Fos/ c-Jun on the Cyclin D1 promoter, and the vascular smooth muscle cells are blocked in the G1 phase, and the blood vessel level is further suppressed. Therefore, SIRT1 is an important gene for regulating the proliferation of smooth muscle cells, and the expression of SIRT1 can be used as an inhibiting vascular smooth muscle cell proliferation and neointimal formation, which affects the remodeling of the blood vessel.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R363
本文編號：2386646
[Abstract]:Neointimal formation is closely related to vascular remodeling, mainly including the proliferation, migration, apoptosis and the synthesis, degradation and rearrangement of vascular smooth muscle cells. Hypervascular smooth muscle cell proliferation is an important pathological basis for atherosclerosis, pulmonary hypertension, systemic hypertension, abdominal aortic aneurysm and restenosis after percutaneous transluminal angioplasty. Cell proliferation is mainly regulated by the following three proteins: Cyclin-dependent kinase (CDK), Cyclin (Cyclin), and Cyclin-dependent kinase inhibitor (CKI). CyclinDl was first synthesized in the cell cycle and reached the peak at the middle of G1, and the complex was formed with CDK4/ CDK6 to control the limit of the G1/ S phase transition. CyclinDl mainly has the regulation of transcription level and the degradation pathway of ubiquitination. Under the stimulation of growth factor, a variety of transcription factors such as AP-1 (activator protein-1), NF-B (nar factor-B), SP1 and the like activate Cyclin Dl, and the expression of Cyclin Dl is regulated at the transcription level, wherein the AP-1 has the most control and control. human SIRT1 (SIRTuin 1) is an NAD +-dependent type III histone de-ethylation enzyme, a high degree of conservation is present between species, and the homology with yeast Sir2 (sience information regulator2) in 7 SIRT members is most High. In recent years, it has been found that SIRT1 is capable of deethanizing histone (H3, H4) and various transcription factors and transcription factors, including MyoD, p300, PGC-1, PPAR, NF-B, Ku70, p53, FOXO, E2F1, etc., in embryonic development, tissue differentiation, metabolism regulation, and cell death. It plays an important role in the repair of death, DNA damage and the resistance to stress. It is found that SIRT1 can also directly regulate cell cycle and cell proliferation in a variety of cells. It is therefore envisaged that overexpression of SIRT1 in smooth muscle cells can inhibit cell proliferation, thereby increasing the proliferation of vascular proliferative diseases. In this study, RT-PCR and Western blot were used to detect the presence of SIRT1 in serum-treated vascular smooth muscle cells and the common carotid artery after ligation. The specific SIRT1 transgenic mice were identified by RT-PCR, Southern blot, Western blot and immunohistochemistry. membrane formation, vascular smooth muscle cell proliferation, extracellular matrix (ECM) and thin artery Cell apoptosis. The proliferation and cell cycle of SIRT1 in vitro were detected by flow cytometry with H3-TdR. The effect of SIRT1 on the level of cell cycle protein expression was observed by Western blot and RT-PCR, and the activity of Cyclin D1 in the downstream gene of AP-1 was detected by luciferase reporter system. Effect of two subunits, c-Fos/ c-Jun, on the Cyclin D1 promoter In this study, the expression of SIRT1 in the vascular smooth muscle cells treated with serum was increased, the final expression was significantly decreased, and the expression of SIRT1 in the model of the common carotid artery of the mouse increased with the formation of the neointima. The results show that the SIRT1 transgenic mice can inhibit the formation of neointima caused by the ligature of the common carotid artery and reduce the extracellular matrix, but the apoptosis of the vascular smooth muscle cells There is a significant change. The overexpression of SIRT1 mediated by adenovirus can inhibit the basal level and the proliferation of primary vascular smooth muscle cells induced by the serum, and make the serum-induced primary vascular smooth muscle cell resistance SIRT1 inhibits the expression of CyclinD1 in the G1 phase of the vascular smooth muscle cells, and further studies indicate that SIRT1 binds to c-Fos/ c-Jun in the vascular smooth muscle cells and inhibits its recruitment on the Cyclin D1 promoter, which may be the result of the inhibition of the expression of CyclinD1 by SIRT1 The molecular basis of the molecular basis. Our study found that the expression of SIRT1 in the rat carotid artery ligation model decreased with the formation of neointima, and the expression of Cyclin D1 could also be reduced in SIRT1 in vivo and may be suppressed. In vascular smooth muscle cells, SIRT1 reduces the expression of CyclinD1 by inhibiting the recruitment of c-Fos/ c-Jun on the Cyclin D1 promoter, and the vascular smooth muscle cells are blocked in the G1 phase, and the blood vessel level is further suppressed. Therefore, SIRT1 is an important gene for regulating the proliferation of smooth muscle cells, and the expression of SIRT1 can be used as an inhibiting vascular smooth muscle cell proliferation and neointimal formation, which affects the remodeling of the blood vessel.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R363
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李碧俠;趙芳;任守文;王學(xué)敏;方曉敏;付言峰;涂楓;王金玉;;去乙酰化酶SIRT1在豬不同組織中的表達(dá)規(guī)律性分析[J];南方農(nóng)業(yè)學(xué)報;2012年11期
相關(guān)碩士學(xué)位論文 前5條
1 陳純娟;白藜蘆醇-Sirt1-Foxo1調(diào)控通路對缺氧心肌細(xì)胞的保護(hù)作用[D];汕頭大學(xué);2008年
2 楊揚(yáng);Sirt1對豬前體脂肪細(xì)胞凋亡的影響[D];西北農(nóng)林科技大學(xué);2010年
3 袁媛;FoxO1調(diào)控骨骼肌纖維類型及其轉(zhuǎn)型機(jī)制的研究[D];西北農(nóng)林科技大學(xué);2010年
4 張朝;白藜蘆醇和煙酰胺對豬前體脂肪細(xì)胞凋亡的作用[D];西北農(nóng)林科技大學(xué);2010年
5 寧小敏;miR-196a對豬脂肪細(xì)胞增殖分化的影響[D];西北農(nóng)林科技大學(xué);2010年
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