Smadl蛋白在睾酮促血管新生过程中的作用及分子机制研究
发布时间：2018-11-12 17:17
【摘要】：研究背景心血管疾病(CVD)是导致人类死亡的主要原因。雄激素能影响心血管疾病的发生和发展。然而,对于雄激素替代治疗的效果还存在争议。一些流行病学研究发现血清中睾酮的水平和心血管疾病的发病率,心血管死亡率以及冠状动脉粥样硬化的严重程度呈负相关,与性别无关。此外,近期最大的队列研究显示血清中睾酮浓度处于正常范围的男性,心肌梗死发生率和死亡率明显降低。同样的,对女性心衰患者进行双盲,随机,控制对照的研究发现,睾酮替代治疗明显的提高了心血管的功能。这些研究表明睾酮可能对心血管系统起保护作用,然而睾酮对心血管系统具体的效应以及分子机制需要进一步研究。血管新生是从已经存在的血管中出芽形成新的毛细血管,对伤口愈合和许多疾病过程中的器官再生至关重要。睾酮能增强受损的心血管系统的血管新生能力,促进其修复。成管能力对于血管重构和修复非常重要。Smad1在内皮中高表达,对血管生成过程中具有重要作用。特异性敲除Smad1蛋白的小鼠由于脉管系统缺乏而死于母体子宫内。有研究报道睾酮能促进血管新生。然而,睾酮促进血管新生的机制以及Smad1是否参与其中,目前还没有相关研究。研究目的探究Smad1蛋白介导睾酮促内皮血管生成中的作用机制。方法及结果本次研究中,我们首先用成管实验观察睾酮对脐静脉内皮细胞(HUVECs)成管的影响,结果显示睾酮能增强内皮的成管能力,沉默Smad1蛋白的表达可阻断睾酮的此效应,说明Smad1蛋白在其过程中的重要作用。随后在培养的HUVECs中,我们用Western Blot检测睾酮对Smad1的活化效应。结果显示睾酮能快速的增加内皮Smad1蛋白的磷酸化。接着用细胞膜非通透性睾酮-牛血清白蛋白偶联抗原(T-BSA)处理,观察发现T-BSA同样能快速活化Smad1。用转录抑制剂放线菌素D(ActD)和蛋白翻译抑制剂放线菌酮(CHX)预处理后Smad1的磷酸化水平,结果显示睾酮激活Smad1蛋白的效应不受影响,表明睾酮活化Smad1不经过转录翻译,呈非基因效应。Western Blot检测AR,c-Src和ERK1/2抑制剂预处理后,发现睾酮激活Smad1的效应被阻断了,研究发现睾酮诱导Smad1磷酸化的效应由mAR介导的,其活性由c-Src/ERK1/2级联反应而发挥生物学效益。此外,在内皮细胞小窝(caveolae)抽提物中检测到AR和c-Src蛋白,免疫共沉淀发现AR与caveolin-1和c-Src的相互作用。破坏caveolae结构或沉默caveolin-1表达可阻断睾酮对信号传导激活和Smad1磷酸化的影响,表明在HUVECs细胞膜小窝内存在功能性信号传导模块。最后,c-Src抑制剂和ERK1/2抑制剂逆转了睾酮促进内皮细胞成管的能力。结论睾酮活化Smad1蛋白并且增加HUVECs的成管能力呈非基因效应,受小窝内的AR/c-Src以及下游的ERK1/2共同调节。我们的研究为探究睾酮对血管的效应提供新的视角。
[Abstract]:Background Cardiovascular disease (CVD) is the leading cause of death in humans. Androgen can affect the occurrence and development of cardiovascular disease. However, the effect of androgen replacement therapy remains controversial. Some epidemiological studies have found that serum testosterone levels are negatively correlated with the incidence of cardiovascular disease, cardiovascular mortality and severity of coronary atherosclerosis, but not with sex. In addition, the largest recent cohort study showed a significant decrease in myocardial infarction and mortality in men with normal serum testosterone levels. Similarly, a double-blind, randomized, controlled study of women with heart failure found that testosterone replacement therapy significantly improved cardiovascular function. These studies suggest that testosterone may play a protective role in the cardiovascular system, but the specific effects of testosterone on the cardiovascular system and its molecular mechanisms need to be further studied. Angiogenesis is the formation of new capillaries from existing blood vessels, which is essential for wound healing and organ regeneration in many diseases. Testosterone enhances angiogenesis and repair of the damaged cardiovascular system. The ability of angiogenesis is very important for vascular remodeling and repair. Smad1 is highly expressed in endothelial cells and plays an important role in angiogenesis. Mice that specifically knocked out the Smad1 protein died in the mother's womb due to a lack of vascular system. Testosterone has been reported to promote angiogenesis. However, there are no studies on the mechanism of testosterone promoting angiogenesis and whether Smad1 is involved. Objective to investigate the role of Smad1 protein in testosterone-induced endothelial angiogenesis. Methods and results in this study, we first observed the effect of testosterone on the (HUVECs) tube formation of umbilical vein endothelial cells by tube forming experiment. The results showed that testosterone could enhance the vascular formation ability of endothelial cells, and silencing the expression of Smad1 protein could block the effect of testosterone. It is shown that Smad1 protein plays an important role in its process. Then, in cultured HUVECs, we used Western Blot to detect the activation effect of testosterone on Smad1. The results showed that testosterone could rapidly increase the phosphorylation of Smad1 protein in endothelial cells. Then treated with membrane permeable testosterone and bovine serum albumin coupled antigen (T-BSA), it was observed that T-BSA could also activate Smad1. rapidly. The phosphorylation levels of Smad1 after pretreatment with transcriptional inhibitor actinomycin D (ActD) and protein translation inhibitor actinomycin (CHX) showed that the effect of testosterone on activating Smad1 protein was not affected, indicating that testosterone activated Smad1 without transcriptional translation. After pretreatment with AR,c-Src and ERK1/2 inhibitors,. Western Blot showed that the effect of testosterone on Smad1 activation was blocked. It was found that the effect of Smad1 phosphorylation induced by testosterone was mediated by mAR. Its activity is produced by c-Src/ERK1/2 cascade reaction. In addition, AR and c-Src proteins were detected in the (caveolae) extract of endothelial cell fossa, and the interaction of AR with caveolin-1 and c-Src was found by immunoprecipitation. Disrupting the structure of caveolae or silencing the expression of caveolin-1 could block the effects of testosterone on signal transduction activation and Smad1 phosphorylation, indicating the existence of functional signal transduction modules in the HUVECs cell membrane fossa. Finally, c-Src inhibitors and ERK1/2 inhibitors reversed testosterone's ability to promote endothelial cell tubulogenesis. Conclusion testosterone activates Smad1 protein and increases the tube-forming ability of HUVECs, which is regulated by AR/c-Src in fossa and ERK1/2 downstream. Our research provides a new perspective on the effects of testosterone on blood vessels.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
，
本文编号：2327710
[Abstract]:Background Cardiovascular disease (CVD) is the leading cause of death in humans. Androgen can affect the occurrence and development of cardiovascular disease. However, the effect of androgen replacement therapy remains controversial. Some epidemiological studies have found that serum testosterone levels are negatively correlated with the incidence of cardiovascular disease, cardiovascular mortality and severity of coronary atherosclerosis, but not with sex. In addition, the largest recent cohort study showed a significant decrease in myocardial infarction and mortality in men with normal serum testosterone levels. Similarly, a double-blind, randomized, controlled study of women with heart failure found that testosterone replacement therapy significantly improved cardiovascular function. These studies suggest that testosterone may play a protective role in the cardiovascular system, but the specific effects of testosterone on the cardiovascular system and its molecular mechanisms need to be further studied. Angiogenesis is the formation of new capillaries from existing blood vessels, which is essential for wound healing and organ regeneration in many diseases. Testosterone enhances angiogenesis and repair of the damaged cardiovascular system. The ability of angiogenesis is very important for vascular remodeling and repair. Smad1 is highly expressed in endothelial cells and plays an important role in angiogenesis. Mice that specifically knocked out the Smad1 protein died in the mother's womb due to a lack of vascular system. Testosterone has been reported to promote angiogenesis. However, there are no studies on the mechanism of testosterone promoting angiogenesis and whether Smad1 is involved. Objective to investigate the role of Smad1 protein in testosterone-induced endothelial angiogenesis. Methods and results in this study, we first observed the effect of testosterone on the (HUVECs) tube formation of umbilical vein endothelial cells by tube forming experiment. The results showed that testosterone could enhance the vascular formation ability of endothelial cells, and silencing the expression of Smad1 protein could block the effect of testosterone. It is shown that Smad1 protein plays an important role in its process. Then, in cultured HUVECs, we used Western Blot to detect the activation effect of testosterone on Smad1. The results showed that testosterone could rapidly increase the phosphorylation of Smad1 protein in endothelial cells. Then treated with membrane permeable testosterone and bovine serum albumin coupled antigen (T-BSA), it was observed that T-BSA could also activate Smad1. rapidly. The phosphorylation levels of Smad1 after pretreatment with transcriptional inhibitor actinomycin D (ActD) and protein translation inhibitor actinomycin (CHX) showed that the effect of testosterone on activating Smad1 protein was not affected, indicating that testosterone activated Smad1 without transcriptional translation. After pretreatment with AR,c-Src and ERK1/2 inhibitors,. Western Blot showed that the effect of testosterone on Smad1 activation was blocked. It was found that the effect of Smad1 phosphorylation induced by testosterone was mediated by mAR. Its activity is produced by c-Src/ERK1/2 cascade reaction. In addition, AR and c-Src proteins were detected in the (caveolae) extract of endothelial cell fossa, and the interaction of AR with caveolin-1 and c-Src was found by immunoprecipitation. Disrupting the structure of caveolae or silencing the expression of caveolin-1 could block the effects of testosterone on signal transduction activation and Smad1 phosphorylation, indicating the existence of functional signal transduction modules in the HUVECs cell membrane fossa. Finally, c-Src inhibitors and ERK1/2 inhibitors reversed testosterone's ability to promote endothelial cell tubulogenesis. Conclusion testosterone activates Smad1 protein and increases the tube-forming ability of HUVECs, which is regulated by AR/c-Src in fossa and ERK1/2 downstream. Our research provides a new perspective on the effects of testosterone on blood vessels.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
，
本文编号：2327710
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