白介素-6中和抗体及索拉菲尼对野百合碱诱导大鼠肺动脉高压的作用及其机制

【作者】 李国；

【导师】 陆慰萱； 施举红；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 [背景]肺动脉高压(pulmonary hypertension, PH)是一组以肺循环阻力和肺动脉压力的持续性升高为特征的病理生理综合征；而动脉型肺动脉高压(pulmonary arterial hypertension, PAH)是其常见类型,是严重的具有潜在破坏力的慢性肺循环疾病。近年研究发现炎症和凋亡/增殖失衡在PAH发生发展中起重要作用。白介素-6(interleukin-6, IL-6)作为一种促炎症因子,可引起肺血管重构,促进PAH的进展。本研究拟利用野百合碱诱导的大鼠PAH模型,以IL-6中和抗体阻断IL-6的活性,来观察其在肺血管炎症反应和凋亡机制中的作用。[目的]1.观察IL-6中和抗体对野百合碱(monocrotaline, MCT)诱导大鼠PAH的作用。2.研究IL-6中和抗体对MCT诱导大鼠PAH肺动脉细胞凋亡影响及对肺动脉重构的作用。[方法]1.建立MCT诱导大鼠PAH模型后,腹腔注射一定剂量IL-6中和抗体进行干预。2.观察各组大鼠一般情况,测定肺动脉平均压(mean pulmonary arterial pressure, mPAP)、右室收缩压(Right ventricular systolic pressure, RVSP)等血流动力学指标,计算右室肥厚指数,评价镜下肺血管病理变化、测量肺动脉壁中膜厚度。3.应用原位杂交和免疫组化技术,分别从mRNA和蛋白水平对不同凋亡蛋白进行定位和半定量分析。[结果]1.IL-6中和抗体可明显降低MCT诱导的大鼠PAH水平、减轻肺动脉中膜厚度和镜下病理改变。2.IL-6中和抗体干预MCT诱导的PAH大鼠后,促进了肺动脉内皮细胞和少许平滑肌细胞的凋亡,其中以Fas-L和Caspase-3表达最明显。[结论]IL-6中和抗体除阻断IL-6炎症机制外,还可能通过促进肺血管细胞凋亡来改善MCT诱导PAH大鼠的肺血管重构。[背景]近10余年来前列环素、内皮素受体拮抗剂、磷酸二酯酶抑制剂等新型的降低肺动脉压药物开始在临床上应用,使部分PAH患者的预后和生活质量有了一定的改善,但因作用靶点单一、无法逆转肺血管重构,因此PAH死亡率仍居高不下。索拉菲尼是一种新型多靶点药物,可抑制酪氨酸激酶受体、丝/苏氨酸激酶受体及VEGFR-1、VEGFR-2、VEGFR-3、PEGFR-等,发挥其抗增殖、抑制新生血管生成、促凋亡等作用。本研究拟通过野百合碱诱导PAH大鼠模型,观察索拉菲尼对已形成明显的PAH和肺血管重构的影响,并探讨其可能作用机制。[目的]观察索拉菲尼对野百合碱(monocrotaline, MCT)诱导PAH大鼠的保护作用并探讨其可能作用机制。[方法]1.建立MCT诱导PAH大鼠模型,以索拉菲尼灌胃进行干预。2.观察大鼠一般情况,测定mPAP、RVSP等血流动力学指标,计算RVHI,评价镜下肺血管病理变化,测量血管壁中膜厚度。3.应用免疫组化技术,对不同凋亡蛋白进行定位和半定量分析。[结果]1.索拉菲尼明显降低MCT诱导PAH大鼠肺动脉压水平,减轻肺血管中膜厚度和镜下病理改变。2.在索拉菲尼干预后,大鼠细胞凋亡主要发生于肺动脉内皮细胞和少许平滑肌细胞,以Bax、Fas和Caspase-3表达最明显。[结论]索拉菲尼对MCT诱导PAH大鼠的保护作用可能与其促凋亡、抑制增殖机制有关。更多还原

【Abstract】 [Backgroud]Pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance and pulmonary artery pressure. Of which the most severe one, pulmonary arterial hypertension (PAH) may lead to right heart failure and death promptly. It has been proved recently that inflammatory mechanism and imbalance between proliferation and apoptosis could play an important role in the developement or progression of PAH. Interleukin-6(IL-6), a kind of proinflammatory factor, can induce pulmonary vascular remodeling and promote the development of PAH. To determine the possible mechanisms of IL-6 on PAH, we investigated the effects of IL-6 neutralizing antibody in rats with monocrotaline-induced PAH.[Objective]1. To observe the effects of IL-6 neutralizing antibody on monocrotaline-induced PAH and analyse its possible mechanisms.2. To study the effects of interleukin 6 on the apoptosis of pulmonary vascular cells of MCT-induced PAH in rats.[Methods]1. After successfully building the monocrotaline-induced PAH model in rats, IL-6 neutralizing antibody was adiministered to the rats by peritoneal injection.2. To observe the general conditions of rats, right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) were detected by right heart catheter. Right ventriclular hypertrophy index was caculated. The histogical feature of pulmonary arteries was evaluated and the thickness of the media measured.3. By in situ hybridization and immunohistochemistry techniques, apoptosis proteins were analysed through semiquantitative method at the stage of mRNA and protein.[Results]1. After IL-6 neutralizing antibody was administered, the level of mPAP decreased significantly and the thickness of the media reduced and the histological feature of pulmonary arteries on MCT-induced PAH in rats improved.2. Apoptosis was mostly expressed by pulmonary arterial endothelial cells and a small amount of smooth muscle cells After IL-6 neutralizing antibody was administered, especially the expression of Fas、Fas-L and caspase-3 significantly.[Conclusions]Besides the anti-inflammation effect, IL-6 neutralizing antibody may improve the MCT induced pulmonary vascular remodeling and decrease the pulmonary arterial pressure through induction of cell apoptosis on MCT-induced PAH in rats. [Backgroud]The advent of new type of drugs, such as prostacyclin analogues, phosphodiesterase inhibitors, and endothelin receptor antagonist, bring a significant advance in the treatment of pulmonary arterial hypertension (PAH). However, the limitation of current therapies for PAH is that they are mostly targeting single and can not reverse pulmonary vacular remodeling, and the therapeutic effects of these agents were limited by their short-life lives, expensive and only partlly effectives in some patients. Sorafenib, a new type of multikinase inhibitor and characterized by antiproliferative, antiangiogenic, and proapoptotic mechanism, can inhibit several receptor tyrosine kinases as well as serine/threonine kinase, PDGF receptor and VEGF receptors. We investigated the effect of sorafenib on established moncrotaline induced pulmonary hypertension in rats.[Objective]To observe the effect of sorafenib on monocrotaline-induced PAH in rats and try to analyse its possible mechanisms.[Methods]1. After successfully building the monocrotaline-induced PAH model in rats, sorafenib was administered to the rats by once daily gavage for 14 days.2. The general conditions of rats were observed. Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) were detected by right heart catheter. Right ventriclular hypertrophy index was caculated. The histological feature of pulmonary arteries was evaluated and the thickness of the medium was measured.3. Apoptosis proteins were analysed by immunohistochemistry technique.[Results]3. After sorafenib was administered, the level of mPAP decreased significantly and the thickness of the media reduced and the histological feature of pulmonary arteries on MCT-induced PAH in rats improved.4. Apoptosis was mostly detected in endothelial cells of pulmonary artery and a small amount of smooth muscle cells, apoptotic proteins Fas、Bax and caspase-3 were expressed significantly After sorafenib was administered.[Conclusions]Sorafenib can ameliorate the pulmonary vascular remodeling and decrease the pulmonary arterial pressure markedly and may be depended on its proapoptotic and antiproliferative effects on MCT-induced PAH in rats.更多还原