Wnt通路抑制剂Wnt-C59对病理性心肌肥大治疗作用的研究

发布时间：2018-01-15 22:37

本文关键词：Wnt通路抑制剂Wnt-C59对病理性心肌肥大治疗作用的研究　出处：《第三军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景:心血管疾病严重威胁着当今中国人民健康,随着人们生活质量的提高,其患病率也呈快速上升趋势。病理性心肌肥大是多种心血管疾病的重要病理基础,负荷增加、炎症刺激、激素异常分泌等外界因素均可导致心肌病理性肥大。如高血压使心脏的后负荷增加可导致心肌肥大。病变早期心脏代偿性向心肥大,心肌收缩力增强,使心输出量维持到正常水平。若病理刺激长期存在,心肌肥大持续加重将导致心脏失代偿,出现离心性肥大,心肌细胞凋亡数目增加且逐渐被成纤维细胞取代,心室壁由厚变薄,心肌收缩力下降,最后可导致严重的心律失常和心力衰竭。抑制心肌肥大进程可有效的改善心功能,提高心血管病患者的生活质量。目前的研究认为,致病理性心肌肥大的主要机制有交感神经系统亢进、RAAS系统的激活等。针对这些机制临床上使用的药物主要有:β-受体阻滞剂(β-blocker)、血管紧张素转化酶抑制剂(ACEI)以及血管紧张素受体阻滞剂(ARB)。尽管这些药物对心肌肥大有一定的治疗效果,但其作用效果有限,尚不能完全阻断病理性心肌肥大的发展,能否逆转心肌肥大也存在争议,故新型抗心肌肥大药物亟需开发和应用。近年来,大量研究表明Wnt通路的激活参与了病理性心肌肥大的发生,阻断Wnt通路有可能成为治疗病理性心肌肥大的新靶点。但目前尚未报道有Wnt通路的阻滞剂被用于治疗心肌肥大的研究。Wnt通路有经典通路和非经典通路之分,两条通路均参与了病理性心肌肥大的进程。经典通路主要通过其下游的β-catenin入核来诱导肥大基因的表达,而非经典通路主要通过其下游的信号分子NFAT入核来诱导肥大基因的表达。常用的Wnt通路抑制剂(sFRPs、DDKs)通常只阻断单一经典通路,由于经典和非经典Wnt通路在心肌肥大发生过程中均被激活,我们假设非选择性地抑制这两条信号通路将达到更佳的抗心肌肥大作用。Wnt-C59可以选择性抑制MBOAT家族的Porcupine酶,从而减弱其介导的Wnt蛋白棕榈酰化作用,从而抑制哺乳动物所有Wnt蛋白的激活,所以其具备同时阻断经典和非经典Wnt通路的作用。在抑制肿瘤增殖的实验中,Wnt-C59已经发挥了显著的治疗作用。本研究从细胞和动物实验两个层面研究Wnt-C59对病理性心肌肥大的治疗作用,并通过对经典和非经典Wnt通路中多个信号分子的检测来验证Wnt-C59的阻断效果,进而为Wnt-C59的临床应用奠定理论基础。研究方法:1.a.给予C57/BL6J小鼠实施主动脉缩窄术(TAC)诱导其病理性心肌肥大,使用Wnt-C59进行干预后观察心肌组织蓬乱蛋白(DVL-1)的mRNA表达量、小鼠体重、心功能、心脏质量指数(心脏质量/体重)、心肌细胞横截面积以及心肌细胞凋亡等指标。b.给予大鼠乳鼠心肌细胞(NRVM)血管紧张素II(Ang-II)刺激诱导细胞水平的病理性心肌肥大,使用Wnt-C59干预后观察心肌细胞DVL-1的mRNA表达量、心肌细胞表面积。2.在细胞实验中,用免疫荧光染色观察β-catenin入核,用Q-PCR检测Wnt信号通路与心肌肥大相关的靶基因C-myc和Cyclin-D1的表达量。3.在细胞实验中用免疫荧光染色观察转录因子NFAT入核,用Q-PCR检测心肌肥大关联基因ANF、α-actin、β-MHC的表达量。研究结果:1.Wnt-C59明显减轻由TAC诱导的小鼠心肌肥大,明显减轻Ang-II诱导的NRVM肥大。2.Wnt-C59对TAC诱导的心功能降低、心肌细胞凋亡增加等有显著的拮抗作用。3.外界的致肥大刺激可通过Wnt-β-catenin经典通路以及Wnt/Ca2+非经典通路的激活使心肌细胞发生肥大;Wnt-C59干预可阻断两条通路中关键转录因子β-catenin以及NFAT的入核,从而发挥抑制病理性心肌肥大的作用。结论:Wnt-C59通过抑制经典和非经典Wnt信号通路,阻断心肌细胞内β-catenin和NFAT的入核,下调C-myc、Cylin-D1、ANF、α-actin、β-MHC等肥大关联基因的表达,继而发挥抑制病理性心肌肥大的作用。
[Abstract]:Background: cardiovascular disease is a serious threat to people's health China today, with improved quality of life, its prevalence rate has also increased rapidly. Pathological cardiac hypertrophy is an important pathological basis of cardiovascular diseases, the increase of load, inflammation, abnormal hormone secretion and other external factors can lead to cardiac hypertrophy such as hypertension. The cardiac afterload increase can lead to myocardial hypertrophy. Early compensatory hypertrophy of heart disease heart, enhance myocardial contractility, cardiac output to maintain the normal level. If the pathological stimuli exist for a long time, the heart muscle hypertrophy will lead to aggravate cardiac decompensation, appear eccentric hypertrophy, myocardial cell apoptosis and increase the number of being gradually become fiber cells replaced by thinner ventricular wall, decreased myocardial contractility, finally can lead to severe arrhythmia and heart failure. Inhibition of cardiomyocyte hypertrophy process may have Improve heart function effectively, improve the quality of life of patients with cardiovascular disease. The present study suggests that the main mechanism of cardiac hypertrophy induced by sympathetic nervous system hyperfunction, activation of the RAAS system. The drug for clinical use of these mechanisms are: beta blockers (beta -blocker), angiotensin converting enzyme (ACEI) inhibitors and angiotensin receptor blockers (ARB). Although these drugs have a certain therapeutic effect on myocardial hypertrophy, but the effect is limited, is still not completely blocked the development of cardiac hypertrophy, can reverse cardiac hypertrophy is also controversial, so the development and application of new anti drug to myocardial hypertrophy. In recent years here, a large number of studies show that the activation of Wnt pathway involved in pathological cardiac hypertrophy, blocking Wnt pathway may become a new target for treatment of pathological cardiac hypertrophy. But has not yet been reported with Wnt Channel blockers are used to study the.Wnt pathway for the treatment of cardiac hypertrophy is the classical pathway and non classical pathway points, two pathways are involved in the pathological process of cardiac hypertrophy. The classical pathway mainly through the expression of its downstream beta -catenin into the nucleus to induce hypertrophy gene expression, rather than by the classical pathway through the signal molecule downstream of NFAT into the nucleus to induce hypertrophy gene. Wnt pathway inhibitors commonly used (sFRPs, DDKs) usually only block a single classical pathway, the classical and non classical Wnt pathways were activated in the process of myocardial hypertrophy, we assume that non selective inhibition of this pathway will reach two anti hypertrophic effect of.Wnt-C59 better can selectively inhibit Porcupine enzyme of the MBOAT family, which weakens the Wnt protein palmitoylation effect mediated, thereby inhibiting activation of Wnt protein in all mammals, so the At the same time by blocking the classical and non classical Wnt signaling pathway. In the inhibition of tumor proliferation experiment, Wnt-C59 has played a significant therapeutic effect. The study from two aspects of Wnt-C59 cell and animal experiments on cardiac hypertrophy therapeutic effect, and through the detection of multiple signal molecules of classical and non classical pathway Wnt the verification of Wnt-C59 blocking effect, which lay a theoretical foundation for the clinical application of Wnt-C59. Methods: 1.a. C57/BL6J mice aorta constriction (TAC) and its pathological myocardial hypertrophy induced by Wnt-C59, in use after the intervention of disheveled protein (DVL-1) expression in myocardial tissue of mRNA mice, weight, heart the function, the heart mass index (heart weight / body weight), cardiomyocyte cross-sectional area and myocardial cell apoptosis index.B. treated neonatal rat cardiomyocytes (NRVM) angiotensin (Ang-II) stimulation Zhang Su II The level of induction of cell pathological myocardial hypertrophy, the expression of Wnt-C59 after myocardial cells were observed by DVL-1 mRNA, the cell surface area of.2. in the cell experiments, using immunofluorescence staining to observe the beta -catenin into the nucleus, with the expression of.3. Q-PCR detected Wnt signal pathway associated with cardiac hypertrophy and Cyclin-D1 target gene C-myc with immune in the experiment of fluorescence in cells was observed in the nuclear transcription factor NFAT, Q-PCR was used to detect myocardial hypertrophy associated gene ANF, -actin alpha, -MHC beta expression. Results: 1.Wnt-C59 significantly reduced TAC induced myocardial hypertrophy in mice, significantly reduced NRVM.2.Wnt-C59 and decreased Ang-II induced hypertrophy of heart function induced by TAC and myocardial cell apoptosis the increase has significant antagonistic effects of.3. induced by external stimulation can hypertrophy through Wnt- pathway and activation of Wnt/Ca2+ beta -catenin classic non classical pathway of myocardial cell hypertrophy; Wnt-C59 intervention can block the key transcription factor -catenin beta two pathways and NFAT into the nucleus, inhibit cardiac hypertrophy. Conclusion: Wnt-C59 can inhibit the classical and non classical Wnt signaling pathway, blocking beta -catenin and NFAT in myocardial cells into the nucleus, under Cylin-D1, ANF, C-myc, alpha -actin the expression of -MHC, beta hypertrophy associated genes, and then inhibit the cardiac hypertrophy.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54

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