【摘要】：研究背景心血管疾病(CVD)是導(dǎo)致人類死亡的主要原因。雄激素能影響心血管疾病的發(fā)生和發(fā)展。然而,對于雄激素替代治療的效果還存在爭議。一些流行病學(xué)研究發(fā)現(xiàn)血清中睪酮的水平和心血管疾病的發(fā)病率,心血管死亡率以及冠狀動脈粥樣硬化的嚴(yán)重程度呈負(fù)相關(guān),與性別無關(guān)。此外,近期最大的隊列研究顯示血清中睪酮濃度處于正常范圍的男性,心肌梗死發(fā)生率和死亡率明顯降低。同樣的,對女性心衰患者進(jìn)行雙盲,隨機(jī),控制對照的研究發(fā)現(xiàn),睪酮替代治療明顯的提高了心血管的功能。這些研究表明睪酮可能對心血管系統(tǒng)起保護(hù)作用,然而睪酮對心血管系統(tǒng)具體的效應(yīng)以及分子機(jī)制需要進(jìn)一步研究。血管新生是從已經(jīng)存在的血管中出芽形成新的毛細(xì)血管,對傷口愈合和許多疾病過程中的器官再生至關(guān)重要。睪酮能增強受損的心血管系統(tǒng)的血管新生能力,促進(jìn)其修復(fù)。成管能力對于血管重構(gòu)和修復(fù)非常重要。Smad1在內(nèi)皮中高表達(dá),對血管生成過程中具有重要作用。特異性敲除Smad1蛋白的小鼠由于脈管系統(tǒng)缺乏而死于母體子宮內(nèi)。有研究報道睪酮能促進(jìn)血管新生。然而,睪酮促進(jìn)血管新生的機(jī)制以及Smad1是否參與其中,目前還沒有相關(guān)研究。研究目的探究Smad1蛋白介導(dǎo)睪酮促內(nèi)皮血管生成中的作用機(jī)制。方法及結(jié)果本次研究中,我們首先用成管實驗觀察睪酮對臍靜脈內(nèi)皮細(xì)胞(HUVECs)成管的影響,結(jié)果顯示睪酮能增強內(nèi)皮的成管能力,沉默Smad1蛋白的表達(dá)可阻斷睪酮的此效應(yīng),說明Smad1蛋白在其過程中的重要作用。隨后在培養(yǎng)的HUVECs中,我們用Western Blot檢測睪酮對Smad1的活化效應(yīng)。結(jié)果顯示睪酮能快速的增加內(nèi)皮Smad1蛋白的磷酸化。接著用細(xì)胞膜非通透性睪酮-牛血清白蛋白偶聯(lián)抗原(T-BSA)處理,觀察發(fā)現(xiàn)T-BSA同樣能快速活化Smad1。用轉(zhuǎn)錄抑制劑放線菌素D(ActD)和蛋白翻譯抑制劑放線菌酮(CHX)預(yù)處理后Smad1的磷酸化水平,結(jié)果顯示睪酮激活Smad1蛋白的效應(yīng)不受影響,表明睪酮活化Smad1不經(jīng)過轉(zhuǎn)錄翻譯,呈非基因效應(yīng)。Western Blot檢測AR,c-Src和ERK1/2抑制劑預(yù)處理后,發(fā)現(xiàn)睪酮激活Smad1的效應(yīng)被阻斷了,研究發(fā)現(xiàn)睪酮誘導(dǎo)Smad1磷酸化的效應(yīng)由mAR介導(dǎo)的,其活性由c-Src/ERK1/2級聯(lián)反應(yīng)而發(fā)揮生物學(xué)效益。此外,在內(nèi)皮細(xì)胞小窩(caveolae)抽提物中檢測到AR和c-Src蛋白,免疫共沉淀發(fā)現(xiàn)AR與caveolin-1和c-Src的相互作用。破壞caveolae結(jié)構(gòu)或沉默caveolin-1表達(dá)可阻斷睪酮對信號傳導(dǎo)激活和Smad1磷酸化的影響,表明在HUVECs細(xì)胞膜小窩內(nèi)存在功能性信號傳導(dǎo)模塊。最后,c-Src抑制劑和ERK1/2抑制劑逆轉(zhuǎn)了睪酮促進(jìn)內(nèi)皮細(xì)胞成管的能力。結(jié)論睪酮活化Smad1蛋白并且增加HUVECs的成管能力呈非基因效應(yīng),受小窩內(nèi)的AR/c-Src以及下游的ERK1/2共同調(diào)節(jié)。我們的研究為探究睪酮對血管的效應(yīng)提供新的視角。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54
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本文編號：2327710