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人SIRT1基因cDNA内皮细胞特异转基因鼠系的建立及其对动脉粥样硬化的影响
【作者】 张庆军；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2006， 博士
【摘要】 动脉粥样硬化是严重威胁人类健康的疾病,虽然其发病机制现在尚未阐明,但是大量研究证明内皮细胞功能失常是其发生的重要条件,在有明显的病理变化之前内皮细胞功能失常已经存在。内皮细胞功能失常有诸多表现,其中内皮细胞特异的一氧化氮合酶（eNOS）基因表达下降是其重要标志,很多动脉粥样硬化的危险因素都可以通过降低eNOS基因表达水平导致内皮细胞释放一氧化氮（nitric oxide,NO）减少,使血管处于不正常的紧张状态,促进动脉粥样硬化的发生。许多对心血管疾病有保护作用的因素和心血管疾病的有效干预措施也可通过上调eNOS表达来纠正内皮细胞功能失常,达到预防和治疗动脉粥样硬化及其并发症的目的。因此,继续寻找能够调节内皮细胞eNOS表达水平的基因,并探索其在内皮细胞功能失常和动脉粥样硬化发生中的作用,将有助于进一步阐明动脉粥样硬化发生的机理。SIRT1（Sirtuin 1）是人类的Ⅲ类组蛋白去乙酰化酶,在各种物种之间呈现高度的保守性,甚至在原核生物中也有其同源基因,在人类的七个Ⅲ类去乙酰化酶成员中与酵母Sir2（silence information regulator 2）的同源性最高。近几年的深入研究表明,人的SIRT1及其小鼠的同源基因Sir2α在胚胎发育、组织分化、代谢调节和抵抗不良环境因素损伤方面发挥重要作用。SIRT1是通过与多种重要的转录因子和转录辅助因子相互作用实现其功能的,这些转录因子包括MyoD、p300、PGC-1α、PPARγ、NF-κB、Ku70、p53以及FOXOs等。因为SIRT1在抵抗不良环境因素的损伤方面发挥重要作用,而动脉粥样硬化的发生过程与遗传和环境危险因素对内皮细胞造成损伤密切相关,所以我们认为:在内皮细胞过表达SIRT1可以保护内皮细胞,抑制动脉粥样硬化的发生。为了验证这个假说,我们进行了如下的研究。首先,在研究内皮细胞的模型—人脐静脉内皮细胞（huamn umbilical veinendothelial cell,HUVEC）检测到了SIRT1的表达;在氧化型低密度脂蛋白（oxidizedlow density lipoprotein,oxLDL）作用后HUVEC的SIRT1表达水平上升,提示SIRT1与oxLDL损伤HUVEC的过程有关。为了研究SIRT1在体内的功能,将SIRT1 cDNA与VE-Cadherin启动子连接,通过显微注射受精卵的方法制备了内皮细胞特异表达SIRT1的转基因小鼠,经过RT-PCR、Western blot和免疫荧光三种方法证明SIRT1在小鼠的血管内皮细胞特异性高表达。为了检测转基因小鼠发生动脉粥样硬化程度的变化,将转基因小鼠与动脉粥样硬化的模型—apoE-/-小鼠杂交,得到基因型为SIRT1+/apoE-/-的小鼠,以基因型为SIRT1-/apoE-/-的同窝同性别鼠作为对照,随机分组喂高脂饮食诱发动脉粥样硬化,持续10周和20周后,检测主动脉粥样斑块的面积变化。结果显示,在喂高脂饮食10周的雄性鼠中,阳性鼠动脉粥样硬化斑块的面积比对照鼠减少（动脉粥样斑块占主动脉总面积的平均值从19.3%减少到13.6%,p＜0.05）。而在喂高脂饮食10周的雌性鼠中,阳性鼠与对照鼠相比动脉粥样硬化的发生没有明显变化,与在研究其他基因时发生的情况相似,表明雌性鼠不适宜作为心血管疾病的动物模型,所以在本研究中分析机制时只针对雄性鼠。在喂20周高脂饮食的鼠中,阳性鼠与对照鼠动脉粥样硬化的程度也没有差异,说明SIRT1可能在动脉粥样硬化发生的早期有保护作用,但这种保护作用可能不足以对抗不良因素长期作用（如高脂饮食20周）造成的损伤。分析动脉粥样硬化发生减少的机制时发现,阳性鼠与对照鼠相比体重、血脂和血糖水平这些系统性因素都没有变化,因此推测内皮细胞本身的因素变化起到了抑制动脉粥样硬化发生的作用,最有可能的是内皮细胞功能失常的改善。因为eNOS表达水平是内皮细胞功能的重要指标,eNOS水平下降是内皮细胞功能受损的标志,而eNOS水平上升是内皮细胞功能改善的指标,于是首先检测eNOS水平的变化,RT-PCR和免疫荧光的结果都显示在阳性鼠内皮细胞中eNOS水平上升。进一步用荧光素酶报告基因系统检测SIRT1对eNOS启动子活性的影响,发现SIRT1共表达可以上调eNOS启动子活性,SIRT1的激活剂Resveratrol也可激活eNOS启动子的活性,而且呈现剂量-效应关系。进一步研究发现FOXO3a可以增加eNOS启动子的活性,而且与SIRT1有协同作用,表明SIRT1上调eNOS表达可能是通过与FOXO3a相互作用实现的。综合上述结果可以得出结论:SIRT1可以通过上调eNOS基因表达改善内皮细胞功能失常而抑制动脉粥样硬化的发生。
【Abstract】 Atherosclerosis is the leading cause of death in western society and recently in China. Although studied thoroughly for many years,the mechanisms of atherogenesis are largely unknown.Among many factors,endothelial cell dysfunction（ECD） plays an important role in atherogenesis.Normal endothelial cells express endothelial cell specific nitric oxide synthase（eNOS） to sustain proper tone of blood vessel.Proper expression level of eNOS is the marker of normal endothelial cell function.Many risk factors of atherosclerosis damage endothelial cells at least in part through repressing eNOS expression.On the other hand,atheroprotective factors and many treatment methods can repress atherogenesis through up regulating eNOS and reverse ECD.Looking for more genes that can regulate eNOS expression and exploring their roles in ECD and atherosclerosis would be helpful in elucidating the mechanisms of atherosclerosis.SIRT1（Sirtuin 1） is one of the seven members of human classⅢhistone deacetylase （HDAC） and is the ortholog of yeast Sir2.SIRT1 gene is highly conserved across species; even in bacteria there exist homologs of SIRT1.Recent studies show that human SIRT1 and its homolog in mice（Sir2α） play very important roles in embryonic development, differentiation,metabolic regulation and stress resistance.SIRT1 exerts these effects through interacting with and regulating the function of many important transcription factors and transcription coactivators such as MyoD,p300,PGC-1α,PPARγ,NF-κB,Ku70, p53,FOXOs and so on.Since SIRT1 plays important roles in stress resistance and atherosclerosis is related to endothelial cell injury caused by environmental and genetic hazard factors,we develop our hypothesis that SIRT1 overexpression in endothelial cells can repress atherogenesis through protection of endothelial cells from damage.We carried out the following experiment to test the hypothesis.In a good model of endothelial cell function research,we observed relative high level of SIRT1 expression in human umbilical vein endothelial cells（HUVEC） and the expression was up regulated when HUVEC was treated with oxidized low density lipoprotein（oxLDL）.This suggested that SIRT1 is related to the process of oxLDL damaging HUVEC.To explore SIRT1’s function in vivo,we created transgenic mouse lines in which SIRT1 is expressed specifically in blood vessel endothelial cells.Reverse transcription polymerase chain reaction（RT-PCR）,Western blot and immunofluorescence show that SIRT1 was properly expressed in endothelial cells of blood vessel.Breeding SIRT1+ mice with apoE-/- mice we obtain SIRT1+/apoE-/- mice and fed them with high fat diet to assess SIRT1’s effect on atherosclerosis.After 10 weeks of high fat diet,male SIRT1+/apoE-/- mice had less atherosclerotic plaques than those of SIRT1-/apoE-/- littermates(the ratio of atherosclerotic plaque to the whole aortic face,13.6%in SIRT1+/apoE-/- compared to 19.3%in SIRT1-/apoE-/- p＜0.05),while no significant change was observed between SIRT1+/apoE-/- and SIRT1-/apoE-/- mice in female mice.Since former researches have shown that female mice are not suitable for cardiovascular disease study,in the next mechanism analysis we use male mice only.No significant difference was observed between positive mice and control mice fed with high fat diet for 20 weeks; this suggested that SIRT1 can only exert atheroprotective effect in early stage of atherogenesis.Systemic factors such as body weight,blood lipid level and blood glucose level remain unchanged between SIRT1+/apoE-/- and SIRT1-/apoE-/- mice,this suggested that there must be some changes in endothelial cells that repressed atherosclerosis.Since eNOS level is the marker of normal endothelial cell function,we detected the change of eNOS expression in these mice.RT-PCR and inmmunofluorescence showed that eNOS was increased in SIRT1+/apoE-/- mice.Next we investigated whether SIRT1 can regulate eNOS transcription.Luciferase reporter test showed that SIRT1 can up regulate the activity of eNOS promoter.SIRT1 activitor,Resveratrol,can also activate eNOS promoter activity in a dose-dependent manner.Further,SIRT1 can activate eNOS promoter synergistically wity FOXO3a.We conclude that SIRT1 exerts its atheroprotective role in endothelial cells through up regulating eNOS expression to reverse endothelial cell dysfunction.