【作者】 于军；

【导师】 朱妙章；

【作者基本信息】 中国人民解放军第四军医大学 ， 生理学， 2003， 博士

【摘要】 背景和目的 钠尿肽(natriuretic peptides,NPs)是一组具有利钠、利尿、舒血管和抗细胞增殖效应的多肽，具有抗心力衰竭、心肌肥厚和移植血管再狭窄的重要作用。钠尿肽受体(natriuretic peptide receptor,NPR)可分为鸟苷酸环化酶(guanyl cyclase，GC)耦联受体和非耦联受体。其中A、B受体亚型(NPR-A，NPR-B)是GC耦联受体，介导NPs发挥重要的生物学功能；而C受体亚型不与GC耦联，属于清除受体，主要影响体内NPs的半衰期。 血管钠肽(vasonatrin peptide,VNP)是人工设计、合成的钠尿肽家族新成员，是心房钠尿肽(atrial natriuretic peptide,ANP)和C-型钠尿肽的嵌合体。已证实，VNP可有效地防治大鼠低氧性肺动脉高压和右心室肥厚，但其作用机制还不明了。NPR-C是直接影响NPs功能的重要因素，低氧时NPR-C水平的变化仍存在争议，VNP与NPR-C的关系也尚未见报道。内皮素-1(endothelin-1,ET-1)是机体缺血、缺氧时，血管内皮、平滑肌、心肌以及心成纤维细胞合成与释放的一种效应很强的缩血管多肽，能引起冠状动脉强烈收缩，造成心肌缺血，甚至坏死。ET-1还促进冠脉平滑肌增生，使管腔狭窄，进一步加重心肌缺氧。VNP对低氧心脏ET-1水平的影响如何，还不十分清楚。本研究首次应用慢性低氧大鼠模型，初步探讨VNP对低氧大鼠右心室NPR-C和ET-1的影响。 人乳内动脉(human internal mammary artery,HIMA)是心脏搭 宽四军区大学博士学位论大桥手术的主要供体血管之一。临床资料表明，移植后血管的痉挛程度是影响手术疗效的重要原因。己发现，VNP对大鼠主动脉和肺动脉具有显著的舒张作用，而VN’I，对HIMA的舒张作用还未见报道。本研究还首次探讨VN’P对HIMA的舒张作用及机制。为开发VN’P成为治疗冠脉搭桥术后血管痉挛的药物提供理论依据。 方 法 ＊）采用减压低氧的方法，模拟 5，500 m的高原低氧环境，饲 养低氧大鼠。对照大鼠在常压、常氧的条件下饲养。 O） 分别用定量PCR和原位杂交的方法分析右心室NPR（与 ETI 的InRNA水平，并用放射免疫测定法测量右心室 ET－1 蛋白含量的变化。同时也观察血浆ANP、右心室 CGMP水平的变化。 o） 采用离体血管灌流的方法，观察VNP对内皮完整和去内 皮HIMA的舒张作用，以及8－Br－cGW、NPR－A，B选择 性抑制剂HS1 八、鸟着酸环化酶选择性抑制剂美蓝 （methylene blue，MB）和＊”激活 K十通道（CaZ”－activated 矿山annel，KC＊的选择性抑制剂hA对这一过程的影响。 门 采用全细胞膜片钳技术观察VN’I，对大鼠肠系膜动脉血管 平滑肌（vascular smooth muscle cells，VSMCs）KC。的作用， 以及8Er七GMP、HS八 ＊和MB对这一过程的影响。 结 果 （1）可氧7～28 d心脏N’P又C InRNInRNA水平显著升高（P＜0＊5）， 一 5——Mp 2003 第四军区大学协士学位论X一 并呈现时间依赖性。每日腹腔注射 50～75 pg／kg的 VNP可 剂量依赖性地减弱低氧心脏NP＊－C的表达（P＜0刀5）。血浆 ANP浓度在低氧 28 d后显著升高（P＜0．05），VNP（75卜g／kg） 使AN P浓度进一步升高（P＜0刀1）。 O）VNP门5 pg／kg）使低氧诱导的心脏ET1 的水平显 著降低（P＜0刀5）。CGMP基础水平未见显著性差异，VNP （7 pg／kg）使低氧心脏的CGMP7k平略高于相应的常氧组， 但无显著性差异（P＞0刀5）。 （3）VNP（10”口一106mo凡）可剂量依赖性地舒张HIMA，该作 用无内皮依赖性。吕一Br－cGMP （l”’～10－’mo讥）可模拟 VNP 的血管舒张效应。HS－142一l（2 x 10”5 mo凡）或 MB（10－’ moUL）完全阻断 VNP舒张作用。TEA门“‘。of／L）减弱 VNP的舒血管作用。 ＊）VNP显著增强大鼠肠系膜动脉VSMCS的KcyVNP的这 一效应可以由8－Br－CGMP模拟，而被HS－142J或MB完 全阻断。 结 论 ＊）VNP抑制慢性低氧导致大鼠右心室NPR七时间依赖性的表 达增加，由此促进血浆内源性ANP水平进一步增加。VNP 还抑制慢性低氧导致大鼠右心室ET．l的表达增加，此过程 与细胞内。GMP水平的变化没有相关性。结果提示，抑制 NPR－C（清除受体）进而增加 NPs的活性和减弱 ET－l的表 达可能是VNP抗慢性低氧作用的重要机制。 一 6——Mp 2003 第四不巨大学博士学位论又 o）VNP对H例具有不依赖内皮的舒张作用，其作用主要是 通过作用于NPR－A，B，即钠尿肽GC受体，引起细胞内的 CGMP水平升高实现的。Kc。可能是其舒张作用的重要效应 分子。 该结果为应用VNP防治慢性低氧性心脏重构和血管移 植术后的痉挛提供了重要的更多还原

【Abstract】 Natriuretic peptides (NPs) are a family of polypeptides possessing natriuretic, diuretic, vasodilative and anti-proliferation actions. In recent years, increasing attention has been paid to their inhibitory effects on heart failure, cardiac hypertrophy and restenosis of transplanted blood vessels in both basic research and clinical application. Natriuretic peptides receptor-A and B (NPR-A, B), which are membrane bound guanylate cyclase coupled to the production of 3’, 5’ cyclic guanosine monophophate (cGMP), mediate the biological actions of NPs, while natriuretic peptides receptor-C (NPR-C) uncoupled with cGMP acts as a clearance receptor by binding with and internalizing NPs.Vasonatrin peptide (VNP) is a new man-made member of natriuretic peptide family, It is a chimera from atrial natriuretic peptide and C-type natriuretic peptide. Recent studies have demonstrated that VNP is effective in preventing and treating hypoxic pulmonary hypertension and right ventricular hypertrophy, however the mechanism is not well understood. NPR-C plays an important role inMay, 2003the regulation of the activity of NPs. The expression of NPR-C in hypoxic heart remains controversial, and the relationship between VNP and NPR-C is still unknown. During hypoxia, ET-1 is strongly synthesized and released by endothelial cells, vascular smooth muscle cells(VSMCs), cardiac myocytes and cardiac fibroblasts. It induces strong contraction and spasm of coronary artery, and results in myocardial ischemia and even necrosis. ET-1 also promotes proliferation of VSMCs, which induces stenosis of coronary artery and more severe hypoxia. However, it is not clear whether VNP affects hypoxia-stimulated increase of ET-1. Therefore, the purpose of this study is firstly to determine the effect of VNP on the expression of NPR-C and ET-1 in chronic hypoxic rats.The human internal mammary artery (HIMA) acts as the donor for coronary artery bypass grafting. Clinical data demonstrate that the spasm of transplanted blood vessel is an important factor reducing the curative effect of coronary artery bypass grafting. It has been found that VNP has strong relaxing effect on rat aorta and pulmonary artery, but there is little report about its effect on HIMA. So the next project is to investigate the vasorelaxing effect of VNP on HIMA and reveal its underlying mechanism.Methods(1) Hypoxia on the elevation of 5,500m was mimicked with low atmospheric pressure.(2) The mRNA level of NPR-C and ET-1 in right ventricle was?9 ? May, 2003detected by the means of quantitative PCR and in situ hybridization respectively. Radioimmunoassay was used to determine the protein level of ET-1. It was also used to measure the plasma concentration of ANP and the level of cGMP in right ventricle.(3) The vasorelaxing effect of VNP on HIMA with and without endothelium was observed by perfusion in vitro. Effects of 8-Br-cGMP, HS-142-1, the selective inhibitor of NPR-A and B, methylene blue (MB), the selective inhibitor of GC, and tetraethylammonium (TEA), the selective inhibitor of Ca2+-activated K+ channel (Kca), were also investigated.(4) The effect of VNP on Kca in VSMCs from rat mesentery arteries was detected by whole-cell patch clamp technique.Results(1) Hypoxia for 7~28 d significantly increased NPR-C mRNA of right ventricle in a time dependent manner (.P<0.05). VNP (50-75 #g/kg, i.p.) significantly reduced the NPR-C mRNA level in a dose dependent manner (PO.05). The plasma concentration of ANP in hypoxic (28 d) rats was significantly higher than that in normoxic group (P<0.05), and VNP at 75 #g/kg made it more higher (P<0.01).(2) VNP at 75 #g/kg significantly inhibited the increase of ET-1 and its mRNA in hypoxic rat hearts (PO.05). Compared with the corresponding normoxic group, the level of cGMP induced by VNP was slightly increased (P>0.05).?10 ? May, 2003(3) VNP from 10-10 to 10-6 mol/L induced a concentr更多还原