【作者】 彭慧茹；

【导师】 吴赛珠；

【作者基本信息】 南方医科大学 ， 老年医学， 2008， 硕士

【摘要】 背景和目的:随着年龄的增长,血管壁的结构和功能会发生重大变化,包括血管腔扩大,血管内膜和中层增厚,血管僵硬度增加,部分血管壁细胞衰老,还有代表血管内皮功能的内皮依赖性血管舒张功能减弱,这些变化改变了各种心脑血管疾病的病理生理机制,从而改变了疾病发生的阈值、严重程度和预后,可能是多种增龄相关性疾病,包括动脉粥样硬化（atherosclerosis,AS）形成的共同机制之一。血管内皮细胞位于血管腔内壁,维持着血管内皮的完整及其功能的正常。血管内皮不仅具有屏障功能,维持血流的通畅,还具有重要的内分泌功能,它可以通过膜受体感知血流动力学和血源性信号的变化,合成并分泌多种生物活性物质,对维护血管结构和功能的正常和凝血系统的稳定起着重要的作用。血管内皮功能障碍与诸多疾病的发生、发展密切相关,目前认为它是AS形成初始阶段的重要改变。近年来的研究表明,体外培养的衰老的血管内皮细胞会发生形态与功能的改变,表现为细胞肥大扁平,内源性一氧化氮分泌减少,呈现出促炎与促栓的表型,这或许在增龄相关的血栓栓塞性疾病中起着重要作用。氧化应激是经典的衰老机制学说之一。以往研究认为,活性氧簇（reactiveoxygen species,ROS）通过氧化应激导致细胞DNA损伤,DNA修复失代偿,引发一系列基因与蛋白的表达异常,细胞发生衰老。在AS发病机制研究中发现,ROS诱导一些炎症因子基因的表达独自或联合参与了AS的形成过程。随着年龄的增长,这些因素在血管壁所造成的损伤不断积累,很可能成为诱导血管衰老发生与发展的最重要因素之一,从而使老年人更容易发生AS。在增龄过程中,除了血管衰老本身因素之外,老年人易发AS的另一个具有老年人群特点的危险因素是体内随年龄增长而发生的性激素平衡的改变。在增龄过程中,体内性激素发生相应变化,其中性激素水平在更年期后发生的变化显示出性激素对机体作用的重要性。流行病学研究表明,男性40岁或50岁后,体内睾酮水平明显降低,而且,男性内源性睾酮水平与多种老年性疾病密切相关。男性内源性睾酮水平与男性血管结构随年龄增长发生的变化之间有无一定的联系呢?目前研究还较少。睾酮对血管内皮细胞衰老的影响,目前还没有文献报道,其作用机制也还没有明确。因此,我们使用低浓度过氧化氢（hydrogen peroxide,H₂O₂）诱导人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）衰老,通过观察测定HUVECs在ROS的作用下各种衰老指标（细胞增殖能力、β-半乳糖苷酶的表达）、细胞内氧化应激水平以及细胞周期调控因子（去磷酸化Rb蛋白）的表达变化,探讨HUVECs衰老的机制,并予睾酮及性激素受体拮抗剂干预,探讨不同浓度睾酮对活性氧诱导HUVECs早衰的干预作用及其可能的作用机制,为明确睾酮在干预血管老化中的作用,及其作用机制提供新的理论和实验依据,或许能为增龄相关脉管系统疾病的防治提供新的靶点。方法:1、培养HUVECs,采用第4～6代细胞。使用60μmol/L H₂O₂作用72 h,诱导HUVECs发生衰老。2、分组:①正常对照组:给予低血清（2%）培养基;②H₂O₂对照组:给予低血清培养基+H₂O₂（60μmol/L）;③睾酮干预组:分别给予睾酮3nmol/L,30nmol/L,300nmol/L,3μmol/L培养30min后,再加予H₂O₂（60μmol/L）;④机制研究组:分别给予ICI182,780、氟他胺1μmol/L培养30min后,给予睾酮（30nmol/L）,30min后再给予H₂O₂（60μmoFL）。或先给予抗氧化剂N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine,NAC）1μmol/L培养30min后,再给予H₂O₂（60μmol/L）。3、MTT法检测细胞增殖能力。细胞染色法检测各组细胞的衰老相关的β-半乳糖苷酶（senescence-associatedβgalactosidase,SA-B-gal）表达情况。2,7-二氯荧光黄（2,7-dichlorofluorescein,DCF）荧光显色检测细胞内活性氧水平。Western Blot法检测去磷酸化Rb蛋白的表达量。结果:1、不同浓度睾酮及生理浓度睾酮合用性激素受体拮抗剂对H₂O₂诱导的HUVECs增殖率的影响:与正常对照组比较,H₂O₂对照组的HUVECs增殖率下降,差异具有显著性（P=0.000）。LSD组间多重比较结果提示,与H₂O₂对照组比较,30nmol/L（生理浓度）睾酮干预组与300nmol/L、3nmol/L（稍高于或低于生理浓度）睾酮干预组的HUVECs的增殖率都具有升高的趋势（P=0.058,P=0.127,P=0.335）;3μmol/L（高于生理浓度）睾酮干预组却具有相反的作用（P=0.000）。与30nmol/L睾酮干预组比较,先给予雌激素受体拮抗剂ICI182,780孵育,可以降低HUVECs的增殖率,差异具有显著性（P=0.000）;而先给予雄激素受体拮抗剂氟他胺,却可以升高HUVECs的增殖率,差异也具有显著性（P=0.000）。2、不同浓度睾酮及生理浓度睾酮合用性激素受体拮抗剂对H₂O₂诱导的HUVECs的SA-B-gal阳性细胞百分率的影响:与正常对照组比较,H₂O₂对照组的HUVECs的SA-β-gal染色阳性细胞百分率升高,差异具有显著性（P=0.000）。LSD组间多重比较结果提示,与H₂O₂对照组比较,30nmol/L（生理浓度）睾酮干预组与300nmol/L、3nmol/L（稍高于或低于生理浓度）睾酮干预组的阳性细胞百分率都有降低的趋势,P分别为0.094、0.115、0.464。与30nmol/L睾酮干预组比较,先给予雌激素受体拮抗剂ICI182,780预处理,阳性细胞百分率升高,差异具有显著性（P=0.000）;而先给予雄激素受体拮抗剂氟他胺预处理,却可以降低阳性细胞百分率,差异也具有显著性（P=0.038）。3、抗氧化剂、生理浓度睾酮及其合用性激素受体拮抗剂对H₂O₂诱导的HUVECs内ROS水平的影响:与正常对照组比较,H₂O₂对照组的HUVECs的相对DCF荧光强度增加,差异具有显著性（P=0.000）。LSD组间多重比较结果提示,与H₂O₂对照组比较,抗氧化剂NAC干预组的HUVECs的相对DCF荧光强度减弱,差异具有显著性（P=0.000）;30nmol/L睾酮干预组的HUVECs的相对DCF荧光强度也呈减弱趋势,P=0.362。与30nmol/L睾酮干预组比较,予雌激素受体拮抗剂ICI182,780预处理,可以增强HUVECs的相对DCF荧光强度,差异具有显著性（P=0.002）;而先予雄激素受体拮抗剂氟他胺预处理,可以减弱HUVECs的相对DCF荧光强度,差异也具有显著性（P=0.004）。4、抗氧化剂、生理浓度睾酮及其合用性激素受体拮抗剂对H₂O₂诱导的HUVECs的去磷酸化Rb蛋白表达的影响:与正常对照组比较,H₂O₂对照组的HUVECs的去磷酸化Rb蛋白表达量增加,差异具有显著性（P=0.000）。LSD组间多重比较结果提示,与H₂O₂对照组比较,NAC干预组的HUVECs的去磷酸化Rb蛋白表达量减少,差异具有显著性（P=0.000）;30nmol/L睾酮干预组的HUVECs的去磷酸化Rb蛋白表达量减少,P=0.132。与30nmol/L睾酮干预组比较,予雌激素受体拮抗剂ICI182,780预处理,细胞的去磷酸化Rb蛋白表达量增多,差异具有显著性（P=0.000）;而予雄激素受体拮抗剂氟他胺预处理,细胞的去磷酸化Rb蛋白表达量表达量减少,差异也具有显著性（P=0.000）。结论:通过本研究,我们能够得出以下结论:1、60Mmol/L的H₂O₂持续作用72h后,HUVECs细胞内活性氧水平增加,去磷酸化Rb蛋白表达增加,细胞增殖能力减弱,SA-β-gal染色阳性细胞百分率增加,细胞发生了衰老。提示ROS引起的氧化应激是HUVECs衰老的重要机制之一。2、30nmol/L（生理浓度）睾酮与300nmol/L、3nmol/L（稍高于或低于生理浓度）睾酮都具有升高H₂O₂诱导的HUVECs的增殖率、降低SA-β-gal染色阳性细胞百分率的趋势。3μmol/L（高于生理浓度）睾酮却具有相反的作用。提示睾酮对HUVECs衰老的干预作用与其浓度相关。3、与30nmol/L睾酮干预组比较,先给予雌激素受体拮抗剂ICI182,780孵育,可以拮抗30nmol/L睾酮的干预作用,增加HUVECs细胞内ROS水平和去磷酸化Rb蛋白表达,降低H₂O₂诱导的HUVECs的增殖率,升高SA-β-gal染色阳性细胞百分率;而先给予雄激素受体拮抗剂,却具有相反的作用。提示生理浓度的睾酮或许能通过部分芳香化转化为雌激素后,作用于雌激素受体,减少细胞内的氧化应激和去磷酸化Rb蛋白表达,影响细胞周期,干预血管内皮细胞衰老。更多还原

【Abstract】 [Objective]The structure and fuction of vessel wall change gravely with advancing age, which includes luminal enlargement, initimal and medial thickening, increased vascular stiffness, partial vascular cells aging, and the endothelium-dependent vasodilator respones which are estimated of endothelial function declines. Age-associated remodeling of the vascular wall changes the pathophysiological of cardiovascular and Cerebrovascular diseases, thus alters the occurrence threshold, the severity and the prognosis of vascular diseases, it may contribute to many age-related diseases, including atherosclerosis （AS ）.The vascular endothelium is situated at the interface between the blood and the vascular wall/tissue and is more than a protective barrier since it possesses anticoagulatory properties and generates a number of autocoids that regulate vascular tone and homeostasis. There is a intimate relationship between endothelium dysfunction and the beginning and progress of many diseases, and endothelium dysfunction exists in the early stage of many diseases. Recently, some research indicated the senescence vascular endothelial cells cultured in vitro exhibit a flattened and enlarged morphology, secrete less endothelial nitrogen monoxidum, and present a phenotype of pro-imflammation and pro-throm, it may contribute to the age-related ahterothrombotic diseases.Oxidative stress is one of the most classic mechanism of aging. Some evidences supported that reactive oxygen species （ ROS ） induce the DNA of cells injured by oxidative stress, when the DNA repair decompensation happen, the express of cell genes change, then cells enter an irreversible growth arrest and being senescence. In the research of the pathogenesis of AS, scientists discovered ROS induce the expression of some inflammation factors which take part in the process of AS independently or jointly. With advancing age, the damage induced by those factors accumulates, it may be one of the most important factors that induces the occurrence and development of vascular aging, thus to make AS more liable to happen in the aged.In the elderly, besides the age-related remodeling of vessel, another risk factor why the AS liable to happen in the aged is the age-related change of sex hormone balance. In the progress of aging, corresponding change happen in endogenous sex hormone balance. Research showed endogenous testosterone level are significantly decreased in men more than 50 years. Is it any relation between the age-related change of endogenous testosterone level and the age-related vascular remodeling in men? At present, there is few research about it, and it is lack that the research about the effects of testosterone on the senescence of vascular endothelial cells and its mechanism of action.So we induced human umbilical vein endothelial cells （ HUVECs ） senescence by hydrogen peroxide （ H₂O₂ ） at the low concentration, then detected a few senescence biomakers of HUVECs, such as the proliferation of cells and senescence associatedβgalactoside （ SA-β-gal ） activity, and the intracellular oxidative stress status and the expression of hypophosphorylated Rb protein （ a cell cycle regulating factor）, and approached the aging mechanism of HUVECs, then intervened with testosterone and sex receptor antagonist, and detected the effects of testosterone on the senescence of HUVECs induced by ROS and approached its possible mechanism of action. So as to identify the effect of testosterone on the pathogeny of vascular aging, and provide new theory and experiment evidence to its mechanism of action, it may provide new target for the prevention and cure of age-related vascular diseases.[Methods]1. Culture HUVECs, the cells of passages of 4-6 were used in the experiments. Inducing HUVECs senescence by 60μmol/L H₂O₂ for 72h.2. Groups: 1) nomal control group: cultured with low serum （ 2% ） medium. 2) H₂O₂ control group: cultured with low serum medium and H₂O₂. 3) testosterone intervention group: cultured with testosterone at different concentrations （ 3nmol/L, 30nmol/L, 300nmol/L, 3μmol/L ） for 30min respectively, then added H₂O₂. 4) mechanism research groups: cultrued with estrogen receptor antagonist ICI182, 780 or androgen receptor antagonist flutamide （ 1μmol/L） for 30min respectively, and added testosterone （ 30nmol/L ） for 30min, then added H₂O₂. Or cultured with antioxygen N-acetyl-L-cysteine （ 1μmol/L） for 30min, then added H₂O₂.3. Detecting the proliferation of cells by MTT method. Detecting the SA-μ-gal activity by cells staining method. And detecting intracellular ROS level by DCF fluorescence coloration. Detecting the hypophosphorylated Rb protein expression by Western blot method[results]1. Effects of testosterone with or without sex receptors antagonists on the proliferation of HUVECs stimulated by H₂O₂.The proliferation of HUVECs in H₂O₂ was lower （ P<0.001 versus nomal control group ）. And the proliferation of HUVEC in the testosterone at low concentration （ 3nmol/L, 30nmol/L and 300nmol/L ） intervention groups was higher, most significantly at the concentration of 30nmol/L （ P=0.335, P=0.058, P=0.127 versus H₂O₂ control group, respectively ）, whereas it was opposite in the testosterone at the higher concentration （ 3μmol/L ） intervention group （ P<0.001 versus H₂O₂ control group ）. The estrogen receptors antagonist ICI182, 170 （ P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ） rather than the androgen antagonist flutamide （ P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ） at the concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.2. Effects of testosterone with or without sex receptors antagonists on the percentage of SA-β-gal staining positive of HUVECs stimulated by H₂O₂.The percentage of SA-β-gal positive cells was higher in H₂O₂ control group （ P<0.001 versus normal control group ）. And the percentage of SA-β-gal positive cells was lower in testosterone at the low concentrations （ 3nmol/L, 30nmol/L and 300nmol/L） intervention groups, most significantly at the concentration of 30nmol/L （ P=0.464, P=0.094, P=0.115 versus H₂O₂ control group, respectively ）, whereas it was opposite in the testosterone at the higher concentration （ 3μmol/L） intervention group （ P<0.001 versus H₂O₂ control group ）. The estrogen receptors antagonist ICI182,170 （ P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ） rather than androgen antagonist flutamide （ P=0.038 versus testosterone at the concentration of 30nmol/L intervention group） at concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L. 3. Effects of testosterone at the physiologic concentration with or without sex receptor antagonist on the intracellular oxidant status of HUVECs stimulated by H₂O₂.The relative DCF fluorescence intensity of H₂O₂ control group was higher （P<0.001 versus nomal control group ）. And the relative DCF fluorescence intensity in the NAC group was lower （ P<0.001 versus H₂O₂ control group ）. And it was lower in testosterone at the concentration of 30nmol/L （physiologic concentration ） intervention group （ P=0.362 versus H₂O₂ control group ）. The estrogen receptor antagonist ICI182,170 （ P = 0.002 versus testosterone at the concentration of 30nmol/L intervention group） rather than androgen antagonist flutamide （ P=0.004 versus testosterone at the concentration of 30nmol/L intervention group ） at the concerntration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.4. Effects of testosterone at the physiologic concentration with or without sex receptor antagonist on the hypophosphorylated Rb protein expression in HUVECs stimulated by H₂O₂.The hypophosphorylated Rb protein expression was more in HUVECs of H₂O₂ control group （ P<0.001 versus nomal control group ）. And it was less in the cells of NAC intervention group （ P<0.001 versus H₂O₂ control group ）. And it was less in the cells of testosterone at the concentration of 30nmol/L （ physiologic concentration ） intervention group （ P=0.132 versus H₂O₂ control group ）. The estrogen receptors antagonist ICI182,780 （ P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ） rather than androgen antagonist flutamide （ P<0.001 versus testosterone at the concentration of 30nmol/L intervention group ） at the concentration of 1μmol/L inhibited the delaying effect induced by testosterone at the concentration of 30nmol/L.[Conclusion]1. Cultured the cells in low concentration of H₂O₂ （ 60μmol/L ） for 72h, increased the ROS level and the expression of hypophosphorylated Rb protein in HUVECs, its proliferation was depressed and the percentage of SA-β-gal staining positive cells was increased significantly vs the nomal control group, cells became senescence. These demonstrated that ROS plays an important role in the process of HUVECs senescence.2. Testosterone at low concentrations （ 3nmol/L, 30nmol/L and 300nmol/L） had the trend of delaying the depression of proliferation and the increase of SA-6-gal staining positive cells percentage of HUVECs stimulated by H₂O₂, whereas higher concentration （3μmol/L） had opposite effect. These demonstrated that testosterone modulate the senescence of HUVECs induced by H₂O₂ in a dose-related manner.3. Estrogen receptor antagonist ICI182,170 rather than androgen antagonist flutamide inhibited the delaying trend induced by testosterone at the concentration of 30nmol/L, such as increased the intracells ROS level and hypophosphorylated Rb expression in HUVECs, and depressed the proliferation and increased the positive cells percentage of SA-6-gal staining of HUVECs stimulated by H₂O₂. We deduced that testosterone at the physiologic concentration may transform to estrogen which acts on estrogen receptor, resulting in decreasing the intracellular oxidative stress and hypophosphorylated Rb protein expression, then finally interfering in the cell cycle and cell senescence.更多还原