Gp130在肺动脉高压发病中的作用机制研究

发布时间：2018-05-30 07:01

本文选题：肺动脉高压 + 糖蛋白130　；参考：《北京协和医学院》2016年博士论文

【摘要】：第一部分：Gp130及其上下游信号通路在肺动脉高压发病中的作用目的：肺动脉高压(Pulmonary Arterial Hypertension, PAH)是一种多因素共同参与的进展性疾病,其发病机制尚不完全清楚。白介素6(Interleukin 6, IL-6)是一种多效应的细胞因子,它是由纤维母细胞、单核/巨噬细胞、T淋巴细胞、B淋巴细胞以及多种肿瘤细胞所产生,IL-6与其受体结合后能够诱导多种细胞的增殖、分化。糖蛋白130(Glycoprotein 130, Gp130)是IL-6细胞内信号转导最为关键的信号分子。本研究旨在探讨Gp130及其上游、下游信号通路在PAH发病中的表达变化,为进一步探索PAH的发病机制提供理论依据。方法：64只SD大鼠,体重240-250g,随机分为生理盐水对照组、野百合碱(Monocrotaline, MCT)1周组、MCT2周组、MCT3周组和MCT4周组。MCT组单次剂量腹腔注射MCT(60 mg/Kg),对照组给予等量生理盐水,建立大鼠PAH模型。在MCT注射后1周、2周、3周和4周,在相应的时间点行右心导管检查测量肺血流动力学指标并处死大鼠。用苏木素-伊红(HE)染色和弹力纤维(VG)染色分析肺血管病理形态学变化。通过免疫组化和免疫印迹技术测定大鼠肺组织IL-6、Gp130和信号转导激活因子3(STAT3)及其下游增殖、凋亡信号的表达变化。结果：与对照组相比,MCT注射后2周,大鼠平均肺动脉压(mPAP)、右室收缩压(RVSP)开始升高,4周时mPAP和RVSP显著升高。肺血管组织病理学分析提示2周时肺中、小动脉中层平滑肌少量增生和血管周围有大量炎症细胞浸润,4周时肺中、小动脉中层平滑肌显著增生和血管周围有较多炎症细胞浸润。MCT组IL-6、Gp130和STAT3表达均较对照组明显升高,Gp130在第1、2周时表达明显增多,第3、4周时表达较前两周减少,但仍显著高于对照组。在第4周时,骨形成蛋白II型受体(Bone morphogenetic protein type Ⅱ receptor, BMPRⅡ)及其下游p-Smad 1/5/8表达明显降低,而它的配体BMP2的表达增高。此外反映肺动脉平滑肌细胞增殖的指标增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)和平滑肌肌动蛋白(a-Smooth muscle actin, a-SMA)表达升高,促增殖血管内皮生长因子(Vascular endothelial growth factor, VEGF)和抗凋亡因子Survivin表达显著增多,促凋亡蛋白Bax和活性Caspase-3的表达也显著升高。结论：MCT诱导的大鼠PAH肺组织中出现中、小动脉平滑肌细胞增生,内皮细胞凋亡。IL-6/Gp130/STAT3信号通路可能参与了PAH的发生和发展。Gp130有望作为干预PAH的潜在治疗靶点。第二部分：Gp130抑制剂干预野百合碱诱导的大鼠肺动脉高压的实验研究目的：肺动脉高压(Pulmonary arterial hypertension, PAH)是以肺动脉平滑肌细胞(Pulmonary arterial smooth muscle cells, PASMCs)失控性增生,管腔狭窄,肺血管重构,肺血管阻力进行性升高为特征的一类疾病。研究表明白介素6(Interleukin 6, IL-6)、信号转导激活因子3(STAT3)在PAH的发生、发展过程中起着重要作用。Gp130是IL-6/STAT3信号通路极为重要的中转站。本研究旨在探讨Gp130抑制剂对PAH肺血管重构的影响以及其在PAH治疗中的作用。方法：96只SD大鼠,体重240g-250g,随机分为生理盐水对照组、MCT组、MCT联合Gp130抑制剂治疗组。Gp130抑制剂组于MCT注射后第14天开始每天腹腔注射5mg/kg的Gp130抑制剂,对照组和MCT组给予等量的生理盐水,持续给药至28天。4周后各组均行右心导管检查,测定肺动脉血流动力学。用HE染色和VG染色分析肺中、小动脉病理形态学变化。通过免疫组化、免疫荧光和免疫印迹技术测定大鼠肺组织IL-6、Gp130和STAT3及其下游增殖、凋亡信号的表达变化。结果：与MCT组比较,Gp130抑制剂干预组降低了MCT大鼠平均肺动脉压(mPAP)和右室收缩压(RVSP),右心室肥厚指数减轻,组织病理学分析显示血管周围炎症细胞减少,肺小动脉中层肥厚程度明显减轻。炎症相关因子IL-6、IL-1β、TNFα及CX3CL1明显下降,血管内皮生长因子(VEGF)和抗凋亡因子Survivin表达显著减少,反映PASMCs增殖的PCNA和a-SMA表达明显降低,促凋亡蛋白Bax和活性Caspase-3的表达也显著减少。此外,BMPRII表达增加,但表达水平低于正常对照组。结论：Gp130抑制剂能够阻断IL-6/Gp130/STAT3信号通路,减轻MCT诱导的大鼠PAH的炎症反应,抑制PASMCS的增殖,逆转肺血管重构。此外,Gp130抑制剂上调MCT大鼠肺组织中BMPRII的表达。因此,Gp130抑制剂有可能成为治疗PAH的一个新的药物。
[Abstract]:Part one: the role of Gp130 and its upstream and downstream signaling pathways in the pathogenesis of pulmonary hypertension: pulmonary arterial hypertension (Pulmonary Arterial Hypertension (PAH)) is a progressive disease involving multiple factors and its pathogenesis is not completely clear. Interleukin 6 (Interleukin 6, IL-6) is a multiple effect cytokine, it is It is produced by fibroblast, monocyte, macrophage, T lymphocyte, B lymphocyte and a variety of tumor cells. The combination of IL-6 and its receptor can induce multiple cell proliferation and differentiation. Glycoprotein 130 (Glycoprotein 130, Gp130) is the most important signal transduction pathway in IL-6 cell signal transduction. This study aims to explore Gp130 and its upstream. The changes in the expression of the downstream signal pathway in the pathogenesis of PAH provide a theoretical basis for further exploring the pathogenesis of PAH. Methods: 64 SD rats and weight 240-250g were randomly divided into normal saline control group, Monocrotaline (MCT) group, MCT2 week group, MCT3 week group and MCT4 week group.MCT group MCT (60 mg/Kg), Rats were given equal amount of saline and established rat PAH model. 1 weeks, 2 weeks, 3 weeks and 4 weeks after MCT injection, right heart catheterization was used to measure pulmonary hemodynamic indexes and death rats at corresponding time points. The pathological changes of pulmonary vessels were analyzed with hematoxylin eosin (HE) staining and elastic fiber (VG) staining. Immunohistochemistry and immuno printing were used. The expression of IL-6, Gp130 and signal transduction activating factor 3 (STAT3) and its downstream proliferation and apoptosis signal expression were measured by trace technique. Results: compared with the control group, the average pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) began to rise at 2 weeks after MCT injection, and the mPAP and RVSP increased significantly at 4 weeks. Pulmonary vascular histopathological analysis suggested At 2 weeks, a small amount of smooth muscle hyperplasia in the middle layer of the pulmonary artery and a large number of inflammatory cells were infiltrated around the blood vessels. At 4 weeks, the significant proliferation of smooth muscle in the middle layer of the lungs and the infiltration of more inflammatory cells around the blood vessels in.MCT group IL-6, the expression of Gp130 and STAT3 were significantly higher than that in the control group. The expression of Gp130 at week 1,2 was significantly increased, and the time table in week 3,4 was observed. At the fourth week, the expression of bone morphogenetic protein II receptor II receptor (Bone morphogenetic protein type II receptor, BMPR II) and its downstream p-Smad 1/5/8 expression significantly decreased, while its ligand BMP2 expression increased at the fourth week. Furthermore, the proliferation of pulmonary artery smooth muscle cell proliferation was also expressed as a proliferating cell nuclear antigen (Pro) Liferating cell nuclear antigen, PCNA) and smooth muscle actin (a-Smooth muscle actin, a-SMA) expression increased, proliferating vascular endothelial growth factor (Vascular endothelial growth) and anti apoptotic factor expression increased significantly, and the expression of apoptotic protein and active protein also increased significantly. Conclusion: In the induction of PAH lung tissue, small artery smooth muscle cell proliferation, endothelial cell apoptosis.IL-6/Gp130/STAT3 signaling pathway may be involved in the development and development of PAH as a potential therapeutic target for PAH intervention. The second part: Gp130 inhibitors interfere with the experimental study of pulmonary hypertension induced by monocrotaline in rats Pulmonary arterial hypertension (PAH) is a kind of disease characterized by uncontrolled proliferation of pulmonary artery smooth muscle cells (Pulmonary arterial smooth muscle cells, PASMCs), stenosis of the lumen, pulmonary vascular remodeling, and increased pulmonary vascular resistance. The study shows that interleukin 6 (Interleukin 6, IL-6), signal transduction activation Factor 3 (STAT3) plays an important role in the occurrence and development of PAH..Gp130 is a very important transfer station of the IL-6/STAT3 signaling pathway. The purpose of this study is to explore the effect of Gp130 inhibitors on the remodeling of PAH pulmonary vessels and its role in the treatment of PAH. Methods: 96 SD rats, weight 240g-250g, were randomly divided into normal saline control, MCT, M. The.Gp130 inhibitor group of the CT combined with Gp130 inhibitor group began to intraperitoneally injected with 5mg/kg Gp130 inhibitors on the fourteenth day after MCT injection. The control group and the MCT group were given the same amount of physiological saline. The right cardiac catheterization was performed for each group after 28 days of.4 week, and the pulmonary artery blood flow mechanics was measured. HE staining and VG staining were used to analyze the lung and small movement. IL-6, Gp130 and STAT3 and its downstream proliferation and apoptosis signal expression were measured by immunohistochemistry, immunofluorescence and Western blot. Results: compared with the MCT group, Gp130 inhibitor intervention group reduced the average pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index in MCT rats. The histopathological analysis showed that the inflammatory cells around the blood vessels were reduced and the level of the middle layer of pulmonary arteriole decreased obviously. The inflammatory related factors IL-6, IL-1 beta, TNF A and CX3CL1 decreased significantly, the expression of vascular endothelial growth factor (VEGF) and anti apoptotic factor Survivin decreased significantly, which reflected the decrease of PCNA and a-SMA expression in PASMCs proliferation and the decrease of the expression of a-SMA. The expression of dead protein Bax and active Caspase-3 also decreased significantly. In addition, the expression of BMPRII increased, but the expression level was lower than that of the normal control group. Conclusion: Gp130 inhibitors can block the IL-6/Gp130/STAT3 signaling pathway, reduce the inflammatory response of PAH in rats induced by MCT, inhibit the proliferation of PASMCS, and reverse the pulmonary vascular remodeling. In addition, Gp130 inhibitor up regulation of MCT. The expression of BMPRII in rat lung tissue, therefore, Gp130 inhibitors may become a new drug for the treatment of PAH.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1

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