SIRT1在血管平滑肌细胞增殖和新生内膜形成中的作用及分子机制研究
发布时间：2018-12-19 08:02
【摘要】：新生内膜形成与血管重塑密切相关,主要包括血管平滑肌细胞的增殖、迁移、凋亡以及基质成分合成、降解及重新排列等过程。过度的血管平滑肌细胞增殖是动脉粥样硬化、肺动脉高压、系统性高血压、腹主动脉瘤及经皮腔内血管成形术后再狭窄等心血管疾病的重要病理基础。细胞增殖主要通过以下三种蛋白进行调控：周期蛋白依赖性蛋白激酶(Cyclin-dependent kinase, CDK),周期蛋白(Cyclin),周期蛋白依赖性蛋白激酶抑制因子(Cyclin-dependent kinase inhibitor, CKI)。其中CyclinDl在细胞周期中最早被合成,于G1中期到达高峰,并与CDK4/CDK6形成复合物,对决定G1/S期转换的限制点进行调控。CyclinDl主要有转录水平及泛素化降解途径的调控,在生长因子刺激下,多种转录因子如：AP-1 (activator protein-1), NF-κB (nuclear factor-κB),SP1等激活CyclinDl,在转录水平调控CyclinDl的表达,其中AP-1对其的调控研究最多。 人类SIRT1 (SIRTuin 1)是NAD+依赖的Ⅲ类组蛋白去乙酰化酶,在各物种之间呈现高度的保守性,在7个SIRT成员中与酵母Sir2 (silence information regulator2)的同源性最高。近年来的研究发现,SIRT1能够去乙酰化组蛋白(H3,H4)和多种转录因子和转录辅助因子,包括MyoD、p300、PGC-1α、PPARγ、NF-κB、Ku70、p53、FOXO、E2F1等,在胚胎发育、组织分化、代谢调节、细胞凋亡、DNA损伤修复和抵抗应激方面发挥重要的作用。在多种细胞中的研究发现,SIRT1还能够直接调节细胞周期和细胞增殖。因此我们设想：在平滑肌细胞中过表达SIRT1能够抑制细胞增殖,从而对抗血管增殖性疾病的增殖。 本研究首先应用RT-PCR和Western blot检测了SIRT1在血清处理的血管平滑肌细胞和结扎后的颈总动脉中的表达变化。随后我们应用RT-PCR、Southern blot、Western blot、免疫组化等方法对平滑肌特异性SIRT1转基因鼠进行鉴定,应用小鼠颈总动脉结扎模型,采用HE染色、免疫组化、Masson染色、TUNEL染色等方法观察转基因鼠新生内膜形成,血管平滑肌细胞增殖,细胞外基质(ECM)及动脉中细胞凋亡。进一步通过H3-TdR、流式细胞术检测SIRT1在体外对细胞增殖和细胞周期的影响。随后,通过Western blot、RT-PCR观察在基础水平和血清诱导下,过表达和干扰SIRT1对细胞周期蛋白表达水平的影响；用荧光素酶报告系统检测对AP-1下游基因CyclinD1活性的影响；ChIP检测SIRT1以及AP-1两个亚基c-Fos/c-Jun在CyclinD1启动子上的募集。 本研究发现血清处理的血管平滑肌细胞中SIRT1表达先升高,最后表达显著降低,小鼠颈总动脉结扎模型中SIRT1表达随新生内膜形成短暂的升高后,显著降低。实验表明SIRT1转基因鼠可以抑制由颈总动脉结扎所引起的新生内膜形成,并且可以减少细胞外基质,但血管平滑肌细胞凋亡没有显著变化。腺病毒介导的SIRT1过表达可以抑制基础水平和血清诱导的原代血管平滑肌细胞增殖,并使其血清诱导的原代血管平滑肌细胞阻滞在G1期。SIRT1在体内外抑制G1期开关分子CyclinD1的表达,进一步研究表明SIRT1在血管平滑肌细胞中结合c-Fos/c-Jun并抑制其在CyclinD1启动子上的募集,这可能是导致SIRT1抑制CyclinD1表达变化的分子基础。 我们的研究发现小鼠颈总动脉结扎模型中SIRT1表达随新生内膜形成降低；SIRT1在体内也可以降低CyclinD1的表达,并可能由此抑制新生内膜形成。在血管平滑肌细胞中,SIRT1通过抑制c-Fos/c-Jun在CyclinD1启动子上的募集,从而降低CyclinD1的表达,使血管平滑肌细胞阻滞在G1期,并进而抑制血管平滑肌细胞的增殖。因此,SIRT1是调控平滑肌细胞增殖的重要基因,提高SIRT1的表达可以作为抑制血管平滑肌细胞增殖和新生内膜形成,影响血管重塑过程的一个新手段。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363
本文编号：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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363
【共引文献】
相关期刊论文 前1条
1 李碧侠;赵芳;任守文;王学敏;方晓敏;付言峰;涂枫;王金玉;;去乙酰化酶SIRT1在猪不同组织中的表达规律性分析[J];南方农业学报;2012年11期
相关硕士学位论文 前5条
1 陈纯娟;白藜芦醇-Sirt1-Foxo1调控通路对缺氧心肌细胞的保护作用[D];汕头大学;2008年
2 杨扬;Sirt1对猪前体脂肪细胞凋亡的影响[D];西北农林科技大学;2010年
3 袁媛;FoxO1调控骨骼肌纤维类型及其转型机制的研究[D];西北农林科技大学;2010年
4 张朝;白藜芦醇和烟酰胺对猪前体脂肪细胞凋亡的作用[D];西北农林科技大学;2010年
5 宁小敏;miR-196a对猪脂肪细胞增殖分化的影响[D];西北农林科技大学;2010年
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