SIRT1在血管平滑肌細胞增殖和新生內膜形成中的作用及分子機制研究
發(fā)布時間：2018-12-19 08:02
【摘要】：新生內膜形成與血管重塑密切相關,主要包括血管平滑肌細胞的增殖、遷移、凋亡以及基質成分合成、降解及重新排列等過程。過度的血管平滑肌細胞增殖是動脈粥樣硬化、肺動脈高壓、系統性高血壓、腹主動脈瘤及經皮腔內血管成形術后再狹窄等心血管疾病的重要病理基礎。細胞增殖主要通過以下三種蛋白進行調控：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在細胞周期中最早被合成,于G1中期到達高峰,并與CDK4/CDK6形成復合物,對決定G1/S期轉換的限制點進行調控。CyclinDl主要有轉錄水平及泛素化降解途徑的調控,在生長因子刺激下,多種轉錄因子如：AP-1 (activator protein-1), NF-κB (nuclear factor-κB),SP1等激活CyclinDl,在轉錄水平調控CyclinDl的表達,其中AP-1對其的調控研究最多。 人類SIRT1 (SIRTuin 1)是NAD+依賴的Ⅲ類組蛋白去乙�；�,在各物種之間呈現高度的保守性,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,SIRT1能夠去乙�；M蛋白(H3,H4)和多種轉錄因子和轉錄輔助因子,包括MyoD、p300、PGC-1α、PPARγ、NF-κB、Ku70、p53、FOXO、E2F1等,在胚胎發(fā)育、組織分化、代謝調節(jié)、細胞凋亡、DNA損傷修復和抵抗應激方面發(fā)揮重要的作用。在多種細胞中的研究發(fā)現,SIRT1還能夠直接調節(jié)細胞周期和細胞增殖。因此我們設想：在平滑肌細胞中過表達SIRT1能夠抑制細胞增殖,從而對抗血管增殖性疾病的增殖。 本研究首先應用RT-PCR和Western blot檢測了SIRT1在血清處理的血管平滑肌細胞和結扎后的頸總動脈中的表達變化。隨后我們應用RT-PCR、Southern blot、Western blot、免疫組化等方法對平滑肌特異性SIRT1轉基因鼠進行鑒定,應用小鼠頸總動脈結扎模型,采用HE染色、免疫組化、Masson染色、TUNEL染色等方法觀察轉基因鼠新生內膜形成,血管平滑肌細胞增殖,細胞外基質(ECM)及動脈中細胞凋亡。進一步通過H3-TdR、流式細胞術檢測SIRT1在體外對細胞增殖和細胞周期的影響。隨后,通過Western blot、RT-PCR觀察在基礎水平和血清誘導下,過表達和干擾SIRT1對細胞周期蛋白表達水平的影響；用熒光素酶報告系統檢測對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)現血清處理的血管平滑肌細胞中SIRT1表達先升高,最后表達顯著降低,小鼠頸總動脈結扎模型中SIRT1表達隨新生內膜形成短暫的升高后,顯著降低。實驗表明SIRT1轉基因鼠可以抑制由頸總動脈結扎所引起的新生內膜形成,并且可以減少細胞外基質,但血管平滑肌細胞凋亡沒有顯著變化。腺病毒介導的SIRT1過表達可以抑制基礎水平和血清誘導的原代血管平滑肌細胞增殖,并使其血清誘導的原代血管平滑肌細胞阻滯在G1期。SIRT1在體內外抑制G1期開關分子CyclinD1的表達,進一步研究表明SIRT1在血管平滑肌細胞中結合c-Fos/c-Jun并抑制其在CyclinD1啟動子上的募集,這可能是導致SIRT1抑制CyclinD1表達變化的分子基礎。 我們的研究發(fā)現小鼠頸總動脈結扎模型中SIRT1表達隨新生內膜形成降低；SIRT1在體內也可以降低CyclinD1的表達,并可能由此抑制新生內膜形成。在血管平滑肌細胞中,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,使血管平滑肌細胞阻滯在G1期,并進而抑制血管平滑肌細胞的增殖。因此,SIRT1是調控平滑肌細胞增殖的重要基因,提高SIRT1的表達可以作為抑制血管平滑肌細胞增殖和新生內膜形成,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.
【學位授予單位】：北京協和醫(yī)學院
【學位級別】：博士
【學位授予年份】：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.
【學位授予單位】：北京協和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R363
【共引文獻】
相關期刊論文 前1條
1 李碧俠;趙芳;任守文;王學敏;方曉敏;付言峰;涂楓;王金玉;;去乙�；窼IRT1在豬不同組織中的表達規(guī)律性分析[J];南方農業(yè)學報;2012年11期
相關碩士學位論文 前5條
1 陳純娟;白藜蘆醇-Sirt1-Foxo1調控通路對缺氧心肌細胞的保護作用[D];汕頭大學;2008年
2 楊揚;Sirt1對豬前體脂肪細胞凋亡的影響[D];西北農林科技大學;2010年
3 袁媛;FoxO1調控骨骼肌纖維類型及其轉型機制的研究[D];西北農林科技大學;2010年
4 張朝;白藜蘆醇和煙酰胺對豬前體脂肪細胞凋亡的作用[D];西北農林科技大學;2010年
5 寧小敏;miR-196a對豬脂肪細胞增殖分化的影響[D];西北農林科技大學;2010年
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