本文選題：肺動脈高壓 + 糖蛋白130�。� 參考：《北京協(xié)和醫(yī)學院》2016年博士論文

【摘要】：第一部分：Gp130及其上下游信號通路在肺動脈高壓發(fā)病中的作用目的：肺動脈高壓(Pulmonary Arterial Hypertension, PAH)是一種多因素共同參與的進展性疾病,其發(fā)病機制尚不完全清楚。白介素6(Interleukin 6, IL-6)是一種多效應的細胞因子,它是由纖維母細胞、單核/巨噬細胞、T淋巴細胞、B淋巴細胞以及多種腫瘤細胞所產(chǎn)生,IL-6與其受體結(jié)合后能夠誘導多種細胞的增殖、分化。糖蛋白130(Glycoprotein 130, Gp130)是IL-6細胞內(nèi)信號轉(zhuǎn)導最為關鍵的信號分子。本研究旨在探討Gp130及其上游、下游信號通路在PAH發(fā)病中的表達變化,為進一步探索PAH的發(fā)病機制提供理論依據(jù)。方法：64只SD大鼠,體重240-250g,隨機分為生理鹽水對照組、野百合堿(Monocrotaline, MCT)1周組、MCT2周組、MCT3周組和MCT4周組。MCT組單次劑量腹腔注射MCT(60 mg/Kg),對照組給予等量生理鹽水,建立大鼠PAH模型。在MCT注射后1周、2周、3周和4周,在相應的時間點行右心導管檢查測量肺血流動力學指標并處死大鼠。用蘇木素-伊紅(HE)染色和彈力纖維(VG)染色分析肺血管病理形態(tài)學變化。通過免疫組化和免疫印跡技術測定大鼠肺組織IL-6、Gp130和信號轉(zhuǎn)導激活因子3(STAT3)及其下游增殖、凋亡信號的表達變化。結(jié)果：與對照組相比,MCT注射后2周,大鼠平均肺動脈壓(mPAP)、右室收縮壓(RVSP)開始升高,4周時mPAP和RVSP顯著升高。肺血管組織病理學分析提示2周時肺中、小動脈中層平滑肌少量增生和血管周圍有大量炎癥細胞浸潤,4周時肺中、小動脈中層平滑肌顯著增生和血管周圍有較多炎癥細胞浸潤。MCT組IL-6、Gp130和STAT3表達均較對照組明顯升高,Gp130在第1、2周時表達明顯增多,第3、4周時表達較前兩周減少,但仍顯著高于對照組。在第4周時,骨形成蛋白II型受體(Bone morphogenetic protein type Ⅱ receptor, BMPRⅡ)及其下游p-Smad 1/5/8表達明顯降低,而它的配體BMP2的表達增高。此外反映肺動脈平滑肌細胞增殖的指標增殖細胞核抗原(Proliferating cell nuclear antigen, PCNA)和平滑肌肌動蛋白(a-Smooth muscle actin, a-SMA)表達升高,促增殖血管內(nèi)皮生長因子(Vascular endothelial growth factor, VEGF)和抗凋亡因子Survivin表達顯著增多,促凋亡蛋白Bax和活性Caspase-3的表達也顯著升高。結(jié)論：MCT誘導的大鼠PAH肺組織中出現(xiàn)中、小動脈平滑肌細胞增生,內(nèi)皮細胞凋亡。IL-6/Gp130/STAT3信號通路可能參與了PAH的發(fā)生和發(fā)展。Gp130有望作為干預PAH的潛在治療靶點。第二部分：Gp130抑制劑干預野百合堿誘導的大鼠肺動脈高壓的實驗研究目的：肺動脈高壓(Pulmonary arterial hypertension, PAH)是以肺動脈平滑肌細胞(Pulmonary arterial smooth muscle cells, PASMCs)失控性增生,管腔狹窄,肺血管重構(gòu),肺血管阻力進行性升高為特征的一類疾病。研究表明白介素6(Interleukin 6, IL-6)、信號轉(zhuǎn)導激活因子3(STAT3)在PAH的發(fā)生、發(fā)展過程中起著重要作用。Gp130是IL-6/STAT3信號通路極為重要的中轉(zhuǎn)站。本研究旨在探討Gp130抑制劑對PAH肺血管重構(gòu)的影響以及其在PAH治療中的作用。方法：96只SD大鼠,體重240g-250g,隨機分為生理鹽水對照組、MCT組、MCT聯(lián)合Gp130抑制劑治療組。Gp130抑制劑組于MCT注射后第14天開始每天腹腔注射5mg/kg的Gp130抑制劑,對照組和MCT組給予等量的生理鹽水,持續(xù)給藥至28天。4周后各組均行右心導管檢查,測定肺動脈血流動力學。用HE染色和VG染色分析肺中、小動脈病理形態(tài)學變化。通過免疫組化、免疫熒光和免疫印跡技術測定大鼠肺組織IL-6、Gp130和STAT3及其下游增殖、凋亡信號的表達變化。結(jié)果：與MCT組比較,Gp130抑制劑干預組降低了MCT大鼠平均肺動脈壓(mPAP)和右室收縮壓(RVSP),右心室肥厚指數(shù)減輕,組織病理學分析顯示血管周圍炎癥細胞減少,肺小動脈中層肥厚程度明顯減輕。炎癥相關因子IL-6、IL-1β、TNFα及CX3CL1明顯下降,血管內(nèi)皮生長因子(VEGF)和抗凋亡因子Survivin表達顯著減少,反映PASMCs增殖的PCNA和a-SMA表達明顯降低,促凋亡蛋白Bax和活性Caspase-3的表達也顯著減少。此外,BMPRII表達增加,但表達水平低于正常對照組。結(jié)論：Gp130抑制劑能夠阻斷IL-6/Gp130/STAT3信號通路,減輕MCT誘導的大鼠PAH的炎癥反應,抑制PASMCS的增殖,逆轉(zhuǎn)肺血管重構(gòu)。此外,Gp130抑制劑上調(diào)MCT大鼠肺組織中BMPRII的表達。因此,Gp130抑制劑有可能成為治療PAH的一個新的藥物。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R544.1

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