【作者】 朱从丽；

【导师】 宋健；

【作者基本信息】 武汉大学 ， 人体解剖与组织胚胎学， 2011， 博士

【摘要】 绝经后或卵巢切除后女性心血管疾病发病率明显增高,这一现象早已被临床和流行病学调查证实,提示卵巢激素(特别是雌激素)对女性心血管系统具有保护效应。但是其机制并未完整建立。在动脉粥样硬化(Atherosclerosis, AS)发生发展过程中,一个不可忽视的现象是,AS病变中存在衰老的内皮细胞和血管平滑肌细胞(Vascular smooth muscle cells, VSMC)。衰老的VSMC异常增高地分泌上皮生长因子、促炎症因子、基质金属蛋白酶、胶原酶等,因而能够刺激邻近细胞增殖、加重炎症反应、破坏局部组织微环境。新近研究显示,因氧化应激、原癌基因激活、端粒缩短等因素导致的VSMC衰老或应激性衰老是加剧局部炎症反应、促进AS病变持续发展的一个重要原因。而雌激素的重要生理功能之一是延缓女性性征器官的衰老并维持其功能。由此推测,雌激素可能通过延缓VSMC的应激性衰老而发挥其心血管保护作用。此外,新近美国Women’s Health Initiative (WHI)组织的一项随机、双盲、对照临床调查分析指出：在接近绝经或绝经早期开始给予雌激素替代治疗,可减少女性冠心病的发病率,但对于绝经晚期或年龄>60岁的妇女,雌激素补充反而导致心血管疾病的危险性增高。由此我们推测,‘雌激素对VSMC的作用可能随细胞来源组织的老龄化程度不同而具有差异。为检验上述假设,本课题从以下三个方面进行了研究：1、雌激素对青壮年大鼠VSMC应激性衰老的影响目的：观察雌激素对青壮年大鼠VSMC氧化应激性衰老的影响。方法：从青壮年(2月龄)雌性未孕SD大鼠主动脉分离的VSMC培养至2-3代,经α-SM肌动蛋白单抗染色阳性细胞率不低于90%时用于实验。上述细胞在有或无最高生理浓度(10-8M),或梯度浓度(10-10～10-8M)17-β-雌二醇(E2)以及雌激素受体(ER)抑制剂ICI182,780(10-SM)存在时,用150μM H202作用2h以诱导应激性衰老。细胞化学染色及流式细胞术分析衰老相关-β-半乳糖苷酶(SA-β-Gal)的胞内定位和衰老(SA-β-Gal阳性)VSMC的比例；免疫荧光标记+流式细胞术分析E2浓度与衰老相关标志蛋白DcR2表达的量效关系。结果：(1)SA-β-Gal细胞化学染色显示：150gM H2O2诱导VSMC 2h后出现大量SA-p-Gal阳性细胞(胞浆呈蓝色,核周染色最深)。典型的衰老细胞体积增大、扁平,胞浆中含较多颗粒或空泡。当存在10-8M E2时,H2O2诱导的SA-β-Gal阳性细胞减少,但仍然高于对照组。H2O2+10-8M E2+10-5M ICI 182,780处理的与单独H2O2处理的比较,阳性细胞量无明显差异。(2)流式细胞术检测SA-β-Gal显示：与对照组比较,单独10-8M E2处理对细胞的衰老率(SA-β-Gal阳性率)没有明显的影响；150μM H202诱导使细胞衰老率显著性升高；当存在10-8M E2时,H202诱导的衰老细胞比例减少(SA-β-Gal阳性细胞率显著下降),但仍高于对照组水平；用10-5M ICI 182,780阻断ER可完全抑制E2的抗氧化应激性衰老的作用。(3)流式细胞术检测DcR2显示：在无E2存在时,150μM H202导致VSMC DcR2的表达显著性升高；梯度浓度(10-10～10-8M)E2的存在呈剂量依赖性地抑制H2O2所诱导的DcR2高表达。用10-5M ICI182,780阻断ER可完全抑制E2的抗H2O2所诱导的DcR2高表达作用。结论：(1)在生理浓度范围内,E2呈剂量依赖性地抑制(但并不能完全阻断)H2O2诱导的青壮年大鼠VSMC早衰,提示E2具有一定的抗VSMC氧化应激性衰老效应。(2)E2抗青壮年大鼠VSMC应激性衰老的作用可被ICI 182,780完全阻断,提示这一效应是通过其受体介导的。2、雌激素抗青壮年大鼠VSMC应激性衰老的机制目的：在第一部分实验中,我们已发现E2具有抗H2O2诱导的青壮年大鼠VSMC应激性衰老的效应。本部分实验试图进一步探讨其可能的分子机制。方法：从青壮年(2月龄)雌性未孕SD大鼠主动脉分离的VSMC培养至2-3代,在有或无10-8M E2存在时,按照上述方法用150μM H2O2诱导VSMC应激性衰老。Pull Down法分析原癌基因Ras活性并用ER阻断实验验证,Western Blot检测p38磷酸化水平及p38调节/活化蛋白激酶(PRAK)、视网膜母细胞瘤蛋白(Rb蛋白)和衰老调控因子(p53,p21,p16)的表达。结果：(1) Pull-Down分析显示：各种处理因素对细胞总Ras表达没有明显影响,150μMH202作用于细胞使GTP-Ras水平较对照组的明显增高,从而导致GTP-Ras/Total-Ras比值显著性增高(Ras活性增高)。10-8M E2的存在可显著降低H2O2诱导的GTP-Ras水平,但并不能纠正到对照组水平。E2的这一作用可被10-5M ICI 182,780完全阻断。(2) Westen Blot分析显示：各种因素处理的细胞总p38表达没有明显变化。在无E2存在时,H2O2处理导致p-p38水平升高、PRAK, p53、p21、p16和Rb蛋白表达增强以及Rb蛋白磷酸化水平下降。当有10-8M E2存在时,上述变化可得到部分纠正,但不能恢复到相应对照水平。结论：(1)E2通过抑制H202激活的原癌基因Ras,降低Ras-p38-PRAK-p53-p21-Rb和Ras-p16-Rb通路的活性而发挥其抗青壮年雌性大鼠VSMC应激性衰老的效应。(2)E2抑制Ras激活的作用是ER依赖性的。3、雌激素对老年大鼠VSMC应激性衰老的影响目的：前两部分实验已经证实E2具有抗H2O2诱导的雌性青壮年大鼠VSMC应激性衰老的作用。以下实验进一步探讨E2的这一效应在雌性老年大鼠VSMC是否有差异?方法：从老年(18月龄)雌性未孕SD大鼠主动脉分离的VSMC培养至第2代,在有或无10-10～10-8M E2存在条件下,用150μM H2O2诱导VSMC应激性衰老。流式细胞术检测SA-β-Gal阳性细胞。为探讨E2抗应激性衰老效应在雌性老年大鼠VSMC出现差异的可能原因,上述实验在有最高生理浓度(10-8M)E2存在条件下,用ER拮抗剂(ICI182,780；10-5M)或E2代谢关键转换酶(CYP450酶)抑制剂ABT(10-5M)进行重复。结果：(1)150μM H2O2诱导使SA-β-Gal阳性细胞率较对照组显著性增高；10-10～10-9ME2的存在对H2O2诱导的VSMC应激性衰老几乎没有影响(SA-β-Gal阳性细胞率在数值上略微下降,但与单独H2O2处理组比较无显著性意义)。但E2浓度达10-8M时,SA-β-Gal阳性细胞率反而显著高于单独H2O2处理组。(2)事先用10-5M ICI182,780阻断ER导致H2O2+10-8M E2处理的VSMC SA-β-Gal阳性细胞率进一步升高。(3)事先用10-5M ABT阻断CYP450酶导致H2O2+10-8M E2处理的VSMC SA-β-Gal阳性细胞率显著下降、甚至低于单独H2O2处理组。结论：(1)E2的抗应激性衰老效应在老年雌性大鼠VSMC几乎完全消失,且当剂量达到最高生理浓度(10-8M)时,E2反而加重老年雌性大鼠VSMC应激性衰老。(2)阻断ER时,10-8M E2加重老年雌性大鼠VSMC应激性衰老的效应进一步增强,提示这种效应并非ER介导,而且可完全掩盖ER介导的抗衰老的作用。(3)阻断CYP450酶时,10-8M E2加重老年雌性大鼠VSMC应激性衰老的效应可被完全抑制,提示高浓度E2加重老年雌性大鼠VSMC应激性衰老的效应很可能是E2代谢产物累积所致。更多还原

【Abstract】 Clinical and Epidemiological investigations have shown that women exhibit significantly higher incidence of cardiovascular disease after menopause or ovariectomy than before menopause, which indicates ovarian hormones, especially the estrogen, possess protective effects in the female’s cardiovascular system, however, the underlying mechanisms of the hormone’s cardiovascular-protective actions are still not fully clear.The unneglected phenomenon is that the senescent vascular smooth muscle cells (VSMCs) and endothelial cells exist in the atherosclerosis (AS) lesion site during the development of it. The senescent VSMCs abnormally secrete epithelial growth factor, pro-inflammatory factor, matrix metalloproteinase, collagenase to stimulate the proliferation of neighboring cells, to increase the inflammatory response, to destroy the local tissue microenvironment. The recent studies have shown that, the senescence or premature senescence of VSMCs caused by oxidative stress, oncogene activation, telomere shortening and so on, is an important reason to increase local inflammation, to promote sustainable development of AS lesion. One of the important physiological actions of estrogen is to delay aging and maintain the functions of female sexuality organs. Therefore, we speculate that estrogen maybe protect the cardiovascular system through delaying VSMC senescence.In addition, the recent analysis for clinical data by American Women’s Health initiative (WHI) showed that:estrogen replacement therapy reduced the incidence of cardiovascular disease among the women closing to menopause or early menopause. However, supplements of estrogen for women at late menopause or more than 60 years old increased the risk of cardiovascular disease. Therefore, we speculate the effects of estrogen on senescence of VSMCs are different because of the different aging degree of tissues. To test these above hypothesises, the researches have been performed from the following three aspects.1. Effects of estrogen on stress-induced premature senescence (SIPS) of vascular smooth muscle cells from young ratsObjectives:To observe the effects of estrogen on H2O2-induced-premature senescence of vascular smooth muscle cells from young rats. Methods:VSMCs of passage 2-3 were cultured from young (2 months) unpregnancy female SD rats’aortas, identified by immunocytochemical staining for a-SM actin, the cells with>90%α-SM actin positive cells were used in the examinations. The VSMCs were stressed by 150μM H2O2 in the presence or absence of the highest physiological concentration of 17-β-estradiol (10-8M E2), or 10-10～10-8M E2 with or without inhibitor of ERs (10-5M ICI182,780). The expression or activation of senescence-associated beta-galactosidase (SA-β-Gal) and senescence-associated marker, DcR2, were detected by cytochemical staining and flow cytometry.Results:(1) SA-β-Gal cell staining showed that:after 150μM H2O2-stress, there were a large number of SA-P-Gal positive cells (blue cytoplasm especially surrounding the nucleus). The typical senescent VSMCs showed an enlarged, flattened shape with granule or vacuolus in the cytoplasm. Pre-administration of the highest physiological concentration (10-8M) of E2 could significantly, but not completely, inhibited the H2O2-induced SIPS. However, this protective effect of E2 was completely blocked by the ER antagonist ICI 182,780 at a concentration of 10-5M.(2) Flow cytometric analysis for SA-β-Gal showed that there was no significant difference in percentages of SA-β-Gal positive cells between the VSMCs treated with 10-8M E2 alone and the control cultures. After 150μM H2O2-stress, the senescent VSMCs increased. Administration of 10-M E2 before stressing significantly inhibited the H2O2-induced SIPS, which was blocked by 10-5M ICI 182,780 completely.(3) Flow cytometric analysis for DcR2 showed that the expression of DcR2 significantly increased after 150μM H2O2-stress. The high level expression of it induced by H2O2 was decreased by pre-administration of 10-10～10-8 M E2 in a dose-dependent manner, which was blocked completely by 10-5M ICI182,780.Conclusions:(1) Physiological concentrations of E2 inhibited, but not completely, H2O2-induced premature senescence of VSMCs from young rats in a dose-dependent manner, which indicates E2 possesses a certain effect on anti-SIPS of VSMCs.(2) The effect of E2 on anti-SIPS of VSMCs could be blocked by ICI 182,780 completely which indicates this effect of E2 is mediated by ERs. 2. Mechanisms of estrogen on anti-SIPS of VSMCs from young ratsObjectives:In the first part examination, we have found that E2 possesses the effect on anti-SIPS of VSMCs from young rats. The following experiments were performed to explore the mechanisms of E2’s anti-SIPS.Methods:VSMCs of passage 2-3 cultured from young (2 months) female SD rats’ aortas were stressed by 150μM H2O2 in the presence or absence of 10-8M E2, with or without 10-5M ICI 182,780. The activation or expression of oncogene Ras, p38, PRAK, p53, p21, p16 and Rb were analyzed through Pull-Down assay or Western Blot.Results:(1) Pull-Down assay revealed that 150μM H2O2-stress caused a sharp increase in GTP-Ras, while not affecting total Ras expression, thus leading to a significant rise in GTP-Ras/Total-Ras ratio. Pre-administration of the highest physiological concentration (10-8M) of E2 could significantly, but not completely, reverse the H2O2-induced Ras activation and this effect of E2 was completely blocked by 10-5M ICI 182,780.(2) Western Blot analysis showed that H2O2-stress led to increases in the level of p-p38, PRAK, p53, p21, p16 and Rb, as well as decrease in the level of p-Rb, and those were significantly inhibited by administration of 10-8M E2 before stressing.Conclusion:(1) Estrogen inhibits SIPS of VSMC from young female rats through suppressing H2O2-induced activation of oncogene Ras to down-regulate both Ras-p38-PRAK-p53-p21-Rb and Ras-p16-Rb pathways.(2) The effect of estrogen on suppressing oncogene Ras’s activation is mediated by ERs.3. Effects of estrogen on SIPS of vascular smooth muscle cells from old ratsObjectives:The experiment results of the first and second parts show that E2 possesses the action on anti-H2O2-induced premature senescence of VSMCs from young rats. The following experiments were performed to investigate whether the action of E2’s anti-SIPS is changed in VSMCs from old female body.Methods:The VSMCs of passage 2 from old (18 months) female SD rats were induced by 150μM H2O2 containing different concentrations of E2 (10～10～10～8M), or 10-8 M E2 plus 10-5M ICI 182,780 or 10-5M ABT (CYP450 inhibitor which blocks transformation of E2 into catecholestradiol. The detections of SA-P-Gal positive cells by flow cytometry were performed with the same methodology for the VSMCs from young rats.Results:(1) Flow cytometric analysis showed that H2O2-stress led to a significantly increased population of SA-β-Gal positive cells (1.85-fold higher than control cultures). But unexpectedly, pre-administration of E2 at 10-10 and 10-9M had no inhibitive effect on H2O2-induced VSMCs senescence; and even more, at its highest physiological concentration (10-8M) of E2 showed an opposite action, increasing H2O2-induced senescence.(2) The senescent-promoting action of E2 at 10-8M was enhanced by 10-5M ICI 182,780.(3) The senescent-promoting action of E2 at 10-8M was eliminated by 10-5M ABT.Conclusion:(1) The effect of E2 on anti-SIPS of VSMCs almost disappears in old rat, and even more, the highest physiological concentration (10-8M) of E2 promotes SIPS of the VSMCs from old rat.(2) The senescent-promoting action of E2 at 10-8M was enhanced by 10-5M ICI 182,780, which indicates this effect is not mediated by ERs and completely covers the effect of E2 on anti-SIPS.(3) The senescent-promoting action of E2 at 10-8M was completely eliminated by 10-5M ABT, which indicates the effect of higher concentration of E2 on promoting SIPS of VSMCs from old rat is mediated by its metabolisms.更多还原