【摘要】：尾加壓素Ⅱ(urotensin Ⅱ，UⅡ)最早是從硬骨魚脊髓尾部分離出來的神經(jīng)環(huán)肽，是迄今為止所知的最強的收縮血管的活性物質(zhì)。UⅡ?qū)ρ艿淖饔糜薪馄蕦W差異和種屬依賴性，并且在不同的生理病理狀態(tài)下對血管的作用不一致。在心血管疾病中，如充血性心力衰竭，心肌梗塞，高血壓，動脈粥樣硬化等疾病中，血漿及局部組織中UⅡ含量升高，并且其它的一些生物活性物質(zhì)，如血管緊張素Ⅱ(angiotensin Ⅱ，Ang Ⅱ)，內(nèi)皮素1(endothelin，ET-1)，，兒茶酚胺類物質(zhì)(catecholamines)含量也升高，說明UⅡ和這些物質(zhì)在疾病的發(fā)生發(fā)展中可能有相關性。目前已有研究報道UⅡ和oLDL、serotonin對平滑肌細胞的增殖具有協(xié)同作用，可以加速高血壓或高脂血癥病人粥樣斑塊的產(chǎn)生，加速疾病的發(fā)展。所以研究UⅡ與其它生物活性物質(zhì)的相互作用，闡明相互作用的機制，對于進一步揭示UⅡ?qū)π难芗膊〉淖饔脵C制及治療心血管疾病新藥開發(fā)與研究都有重要的意義。 本研究采用體外張力測定等方法，觀察UⅡ和Ang Ⅱ?qū)ρ苁湛s的協(xié)同作用。我們采用低劑量不引起明顯的血管收縮的UⅡ和Ang Ⅱ觀察兩者對大鼠胸主動脈(去除內(nèi)皮)的協(xié)同作用，并且探討協(xié)同作用的機制。結果我們發(fā)現(xiàn)2nM、3 nM的Ang Ⅱ和1 nM的UⅡ?qū)ρ艿氖湛s有協(xié)同作用，可以被GPR14阻斷劑urantide和AT_1受體阻斷劑Losartan阻斷，也可以被PKC非特異性阻斷劑chelerythrine阻斷。我們進一步研究發(fā)現(xiàn)在協(xié)同作用的血管中磷酸化的PKCα/βⅡ亞型，磷酸化的MLC和磷酸化的PKC底物含量與對照組相比升高，而單獨加入同等劑量的UⅡ和Ang Ⅱ?qū)τ诹姿峄腜KCα/βⅡ，MLC和PKC底物沒有作用。單獨加入高劑量的UⅡ(10 nM)可以引起血管明顯的收縮，而且可以激活血管中PKCα/βⅡ、PKCθ亞型以及PKC底物和MLC，也可以被GPR14阻斷劑、PKC阻斷劑chelerythrine阻斷，說明PKC參與了UⅡ縮血管的作用，并且在UⅡ縮血管的不同階段，是不同的PKC亞型起作用的。 為了進一步探討PKC在UⅡ收縮血管中的作用，我們用鈣離子阻斷劑(Methoxyverapamil/thapsigargin)阻斷細胞內(nèi)外的鈣離子，觀察到UⅡ?qū)ρ艿氖湛s作用比對照組減少約50％左右，并且可以被PKC阻斷劑chelerythrine阻斷。UⅡ?qū)τ?7周齡自發(fā)性高血壓大鼠(SHR，內(nèi)皮破壞)主動脈的收縮作用強于對照組Wistar-Kyoto(WKY)大鼠，說明UⅡ在不同的病理狀態(tài)下對血管的收縮反應不同，具體機制有待于進一步研究。另外，我們還在人和大鼠的平滑肌細胞上觀察到UⅡ?qū)毎灿惺湛s作用，PKC通路也參與了細胞的收縮反應。 由于UⅡ在心血管疾病中有重要作用，為了觀察UⅡ?qū)π呐K的作用，我們用免疫沉淀方法證實UⅡ受體GPR14(現(xiàn)已被命名為UT受體)蛋白主要存在左 心室，為UⅡ?qū)π呐K的作用提供了直接的證據(jù)。另外，我們還觀察到UⅡ不能引起心肌細胞肥大，可能是由于新生大鼠心肌細胞上UⅡ受體表達較少原因。最后我們構建了含有UⅡ受體GPR14的腺病毒載體，為以后研究GPR14的功能及UⅡ的作用提供基礎。 總之，本實驗首次觀察到UⅡ和Ang Ⅱ通過PKC信號通路的串話對血管收縮有協(xié)同作用，PKCα/βⅡ可能參與了協(xié)同作用，并且不同PKC亞型在UⅡ縮血管的不同時段起不同的作用。UⅡ?qū)HR收縮作用強于WKY。這對于進一步揭示UⅡ在心血管疾病中的作用和其作用的機制將具有十分重要的意義。
[Abstract]:Urotensin II (U II) is the first neurocyclic peptide isolated from the caudal part of the spinal cord of the skeletal fish. It is the most potent vasoconstrictor known to date. The effects of U II on blood vessels are anatomically different and species-dependent, and have different effects on blood vessels under different physiological and pathological conditions. In diseases such as congestive heart failure, myocardial infarction, hypertension, and atherosclerosis, U II levels in plasma and local tissues are elevated, as are other bioactive substances, such as angiotensin II (Ang II), endothelin 1 (ET-1), and catecholamines, said Dr. It has been reported that UII and oLDL, serotonin have synergistic effects on the proliferation of smooth muscle cells, which can accelerate the formation of atherosclerotic plaques in patients with hypertension or hyperlipidemia, and accelerate the development of disease. To clarify the mechanism of interaction is of great significance for further revealing the mechanism of action of UII on cardiovascular diseases and the development and research of new drugs for the treatment of cardiovascular diseases.
In this study, the synergistic effect of U II and Ang II on vasoconstriction was observed by in vitro tension measurement. The synergistic effect of U II and Ang II on thoracic aorta (endothelium removal) in rats was observed by low dose of non-obvious vasoconstriction U II and Ang II, and the mechanism of synergistic effect was explored. UII of nM has synergistic effects on vasoconstriction, which can be blocked by urantide, a GPR14 blocker, and Losartan, a AT1 receptor blocker, or by chelerythrine, a PKC nonspecific blocker. We further investigated the phosphorylated PKC alpha/beta II subtype, phosphorylated MLC, and phosphorylated PKC substrate content in synergistic vessels. Compared with the control group, the phosphorylated PKC alpha/beta II, MLC and PKC substrates did not respond to the same doses of U II and Ang II alone. High doses of U II (10 nM) alone could induce significant vasoconstriction and activate PKC alpha/beta II, PKC theta subtypes, PKC substrates and MLC, and could also be blocked by GPR14, PKC. The blockade of the blocker chelerythrine indicated that PKC participated in the vasoconstriction of UII and played a role in different subtypes of PKC at different stages of UII vasoconstriction.
In order to further explore the role of PKC in U II vasoconstriction, we blocked intracellular and extracellular calcium ions with a calcium blocker (methoxyverapamil/thapsigargin). We observed that the contractile effect of U II on blood vessels was about 50% less than that of the control group, and could be blocked by the PKC blocker chelerythrine. UII could be blocked by a calcium blocker for 17-week-old spontaneous hypertension. The contraction of aorta in rats (SHR, endothelial destruction) was stronger than that in Wistar-Kyoto (WKY) rats, indicating that the contractile response of UII to blood vessels was different under different pathological conditions, and the specific mechanism needed further study. It participates in cell contractile response.
Because U II plays an important role in cardiovascular disease, in order to observe the effect of U II on the heart, we used immunoprecipitation method to confirm that U II receptor GPR14 (now named UT receptor) protein exists mainly in the left side.
In addition, we also observed that U II could not induce cardiomyocyte hypertrophy, possibly due to the low expression of U II receptor in neonatal rat cardiomyocytes. Finally, we constructed an adenovirus vector containing U II receptor GPR14, which could be used to study the function of GPR14 and the effect of U II. For the foundation.
In conclusion, we observed for the first time that U II and Ang II have synergistic effects on vasoconstriction via PKC signaling pathway. PKC alpha/beta II may be involved in the synergistic effect, and different PKC subtypes play different roles in different periods of U II vasoconstriction. The function and the mechanism of its action will be of great significance.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R33

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