Fkbp51基因在心脏发育和心脏纤维化中的作用以及分子机制的探究

发布时间：2018-05-30 22:05

本文选题：FKBP51 + 心脏纤维化　；参考：《北京协和医学院》2017年硕士论文

【摘要】：[目的]心脏纤维化是以心脏间质细胞外基质蛋白积累为特点,造成心脏收缩和舒张功能不全,是许多心脏病理生理变化的前提条件。糖皮质激素(GC)是一类由肾上腺皮质分泌的甾体激素,不但可以通过糖皮质激素受体(GR)调节糖、脂肪和蛋白质的生物合成及代谢,而且糖皮质激素在心血管系统的平衡中也具有重要作用;作为糖皮质激素受体的共伴侣蛋白,FKBP51蛋白参与糖皮质激素受体介导的信号传导过程,因此敲除Fkbp51可能对心脏发育和心脏纤维化的发生产生影响。本论文通过建立Fkbp51基因敲除小鼠心脏纤维化模型,探究FKBP51在心脏发育及心脏纤维化中的作用,为探究心脏发育和研制抗心脏纤维化药物提供理论依据。[方法]利用实验室已有的Fkbp51基因敲除(KO)小鼠,通过建立心脏纤维化模型,分析Fkbp51基因在心脏纤维化过程中的作用。1)小鼠的心脏纤维化模型建立:将实验小鼠分为四组(每组五只),分别为皮下种植缓释泵缓慢释放生理盐水的野生型小鼠(WT-control)、释放生理盐水的Fkbp51基因敲除小鼠(KO-control)、释放异丙肾上腺素(ISO)的野生型小鼠(WT-ISO)、释放异丙肾上腺素的Fkbp51基因敲除小鼠(KO-ISO),持续三周;2)心脏超声与心电功能检测:对以上实验组中小鼠麻醉后,进行超声与心电功能检测;3)心脏表观病理分析:使用电子天平称量实验小鼠心脏重量,使用相机对实验小鼠心脏进行拍照并观察其大小和形态;对各实验组小鼠心脏进行常规石蜡包埋并切片,进行HE染色和Masson染色,探究WT和KO小鼠心脏组织病理学变化;4)利用Real-time PCR技术检测心脏纤维化相关基因mRNA水平上的变化;5)利用免疫组化和免疫荧技术检测心脏纤维化相关基因蛋白水平上的变化;6)血清指标检测。对四组小鼠的血清进行心脏纤维化相关炎症因子和细胞因子验证;7)利用免疫印迹技术检测(1中四组小鼠)心脏组织中纤维化标志性蛋白、TGF-β通路相关蛋白、m-TOR通路相关蛋白的变化。[结果]1)通过免疫组织化学方法鉴定证明成功构建了 WT心脏纤维化动物模型,而Fkbp51KO小鼠并无明显纤维化出现。2)通过超声与心电检测,显示Fkbp51基因敲除后,Fkbp51 KO小鼠主波电压平均水平显著高于WT小鼠且心率显著减慢,表明KO小鼠可能存在房室传导阻滞,且KO小鼠心室肌厚度大于WT小鼠心室肌厚度,心脏发育异常。ISO处理后,超声结果显示,WT小鼠的射血分数、舒张末期左室后壁厚度及左室短轴缩短率均降低,心功能显著降低;而KO小鼠并无显著性变化,心功能相对稳定。心电结果显示WT主波明显变深变高,KO主波变高趋势不如WT明显且心率明显加快,传导也加快,收缩力增强。3)通过对心脏表观进行观察发现:ISO处理组中,WT小鼠心脏明显变大、变重,而Fkbp51 KO心脏无明显改变。4)心脏病理学分析发现,ISO处理后,WT小鼠心脏出现明显肥大,心脏壁变薄,出现大量纤维化;而Fkbp51基因敲除小鼠并未出现纤维化,且心脏壁无明显改变。5)通过Real-time PCR检测,发现ISO处理后Fkbp51基因显著增加;Collagen Ⅰ和AngⅡ在Fkbp51基因敲除小鼠中的表达明显少于野生型小鼠,异丙肾上腺素处理后WT小鼠出现显著性升高,而KO小鼠无明显改变;与心脏纤维化Marker相关的细胞因子TGF-β以及TGF-βR Ⅰ、TGF-βRⅡ也出现此趋势。6)免疫荧光实验,对Collagen Ⅰ蛋白进行验证,发现其在Fkbp51基因敲除小鼠心脏中的表达明显少于野生型小鼠;免疫组化实验,对α-SMA和TIMP1等纤维化标志蛋白进行验证,发现在Fkbp51基因敲除小鼠中的表达明显少于野生型小鼠。7)血清指标检验发现,ISO处理后纤维化标志分子小鼠成纤维细胞生长因子、小鼠超氧化物歧化酶、血管紧张素Ⅱ和小鼠L—乳酸脱氢酶、小鼠肿瘤坏死因子α和小鼠γ干扰素出现显著性改变,而KO小鼠无明显改变。8)免疫印迹实验发现:未经处理时,KO小鼠α-SMA蛋白、P-STAT3蛋白和mTOR蛋白表达增加,异丙肾上腺素处理后WT小鼠FKBP51蛋白表达增加,且CollagenⅠ蛋白和GR蛋白表达也增加。此外KO小鼠比WT小鼠Smad蛋白表达增加,说明TGF-β通路与此过程存在密切相关,此外异丙肾上腺素处理前后,KO小鼠mTOR相关蛋白表达增加,说明mTOR蛋白与此过程密切相关。[结论]FKBP51蛋白通过与糖皮质激素受体相互作用,参与糖皮质激素信号传导,通过TGF-β信号通路和mTOR信号通路调控小鼠心脏发育和由异丙肾上腺素诱导的小鼠心脏纤维化的发生。目的通过分析野生型小鼠(WT)和Fkbp51基因敲除(KO)小鼠分别使用异丙肾上腺素处理和心脏左前降支结扎两种方法构建心脏纤维化模型前后心脏mRNA表达系谱的变化,研究Fkbp51基因在心脏发育和心脏纤维化过程中的作用。方法利用第二代高通量基因测序技术,对异丙肾上腺素处理和心脏左前降支结扎两种方法构建的纤维化模型小鼠的心脏进行mRNA表达谱测序,将测序的数据用DEGseq进行差异分析,限定差异条件后筛选出各组小鼠心脏的差异基因,通过测序数据各组间比较得到组间的基因火山图,进一步探索组间差别并做出各组的热图,之后利用在线工具DAVID对差异基因进行GO本体分析和KEGG通路分析,并利用Genecards对基因进行注释。结果(1)Fkbp51的缺失主要导致与核糖体相关的合成与降解,通过特异性的核糖体的功能变化引起心脏的发育损伤。(2)异丙肾上腺素处理后,WT小鼠呈现出纤维化,而KO小鼠无明显纤维化。WT小鼠处理后PI3K-Akt信号通路出现明显改变,而KO小鼠存在明显改变的是核糖体。(3)左前降支结扎后,WT和KO小鼠两组均存在纤维化,但KO小鼠会有比较高的死亡率。WT小鼠处理后主要影响酶活性剂代谢,KO小鼠主要影响酶活性和离子活性,并一定程度上影响感染相关的通路。(4)KO组ISO处理后,存在显著性改变的依然是核糖体,与Fkbp51基因敲除后的变化一致,这可能是基因敲除后影响心脏发育及ISO处理后抗纤维化的原因。(5)LAD处理后,WT和KO小鼠相比,改变的主要是酶的活性变化,但KO小鼠存在较高的死亡率,可能是由于氧化还原酶的变化及相关疾病的易感性。结论本研究通过对Fkbp51KO与WT小鼠的心脏异丙肾上腺素和左前降支结扎两种方法构建心脏纤维化后的RNAseq数据进行分析,发现Fkbp51基因在心脏发育和心脏纤维化中的作用是一个多基因、多途径、多信号通路相互作用的过程,为下一步深入研究Fkbp51基因在心脏发育和心脏纤维化中作用的分子机制提供了理论基础,进一步为Fkbp51在心脏疾病中可能起到的作用做出理论铺垫。
[Abstract]:[Objective] cardiac fibrosis is characterized by the accumulation of extracellular matrix protein in the cardiac stromal cells, causing cardiac contraction and diastolic dysfunction. It is a prerequisite for many cardiac pathophysiological changes. Glucocorticoid (GC) is a steroid hormone secreted by the adrenal cortex. It can not only regulate sugar, fat, and fat through the glucocorticoid receptor (GR). The biosynthesis and metabolism of proteins and glucocorticoids also play an important role in the balance of the cardiovascular system; as a co chaperone of glucocorticoid receptors, FKBP51 protein is involved in the signaling process mediated by glucocorticoid receptors, so knockout Fkbp51 may have an effect on the development of the heart and the occurrence of cardiac fibrosis. By establishing a model of cardiac fibrosis in Fkbp51 knockout mice, this paper explores the role of FKBP51 in heart development and cardiac fibrosis, and provides a theoretical basis for exploring heart development and developing anti cardiac fibrosis drugs. [Methods] the Fkbp51 gene knockout (KO) mice in the laboratory were used to establish a model of cardiac fibrosis by establishing a model of cardiac fibrosis. The role of Fkbp51 gene in the process of cardiac fibrosis was established in.1) the model of cardiac fibrosis in mice: the experimental mice were divided into four groups (five rats in each group), respectively, the wild type mice (WT-control) that release the physiological saline of the subcutaneous implant sustained-release pump, the release of Fkbp51 gene knockout mice (KO-control) and the release of isoproterenol (I). SO's wild type mice (WT-ISO), release isoproterenol Fkbp51 knockout mice (KO-ISO), last three weeks; 2) cardiac ultrasound and electrocardiogram function test: after anaesthesia in the mice in the experimental group, ultrasonic and electrocardiogram function test; 3) cardiac apparent disease analysis: use electronic balance to weigh the heart weight of mice and use the camera The heart of the experimental mice was photographed and the size and shape of the heart were observed. The heart of the mice in the experimental group was embedded and sliced by paraffin, HE staining and Masson staining were carried out to explore the pathological changes in the heart of WT and KO mice. 4) the changes in the mRNA level of the cardiac fibroblast related genes were detected by Real-time PCR technology; and 5) the use of the immune group. Changes in the level of gene protein related to cardiac fibrosis were detected by chemical and immunofluorescence technology; 6) detection of serum indicators. Test the serum levels of inflammatory factors and cytokines in the serum of four groups of mice; 7) the detection of fibrotic markers in the heart tissues of four groups of mice in 1 of 1 groups, and TGF- beta pathway related proteins by immunoblotting technique The changes in m-TOR pathway related proteins. [result]1) the WT cardiac fibrosis animal model was successfully constructed by immunohistochemical method, while the Fkbp51KO mice had no obvious fibrosis.2). The average level of the main wave voltage of the Fkbp51 KO mice was significantly higher than that of the WT mice after the Fkbp51 gene knockout. The heart rate decreased significantly, indicating that the KO mice might have atrioventricular block, and the thickness of ventricular myocytes in KO mice was greater than that of WT mice. After cardiac dysplasia.ISO treatment, ultrasonic results showed that the ejection fraction of WT mice, the thickness of left ventricular posterior wall and the short axis shortening rate of left ventricle decreased, and the cardiac function decreased significantly in WT mice, and the KO mice did not. The cardiac function was relatively stable. Electrocardiogram showed that the main wave of WT was obviously deeper and higher, the trend of KO main wave was not as obvious as WT and the heart rate was obviously accelerated, the conduction was accelerated, and the contractile force was enhanced.3). By observing the cardiac apparent view, the heart of WT mice was obviously larger and heavier in the ISO treatment group, but the heart of Fkbp51 KO had no obvious change of.4). The heart pathology analysis showed that after ISO treatment, the heart of WT mice showed obvious hypertrophy, the heart wall thinned and a large number of fibrosis appeared, while Fkbp51 gene knockout mice did not appear fibrosis, and the heart wall did not change.5) by Real-time PCR detection, it was found that Fkbp51 gene was significantly increased after ISO treatment; Collagen I and Ang II were in Fkbp51 gene knockout. The expression in mice was significantly less than that in the wild type mice. The WT mice were significantly increased after isoproterenol treatment, but there was no obvious change in the KO mice. The cytokine TGF- beta and TGF- beta R I related to Marker of cardiac fibrosis, and TGF- beta R II also appeared in this trend.6) immunofluorescence experiment, and the Collagen I protein was verified, and it was found F. The expression in the heart of kbp51 gene knockout mice was significantly less than that in the wild type mice; the immunohistochemical test was used to verify the fibrosis markers such as alpha -SMA and TIMP1, and it was found that the expression in the Fkbp51 knockout mice was significantly less than that of the wild type mouse.7) and the serum index test was found after ISO treatment, and the fibrotic fibroblasts of the fibrotic markers were produced after ISO treatment. Long factor, mouse superoxide dismutase, angiotensin II and mouse L - lactate dehydrogenase, mouse tumor necrosis factor - alpha and interferon - gamma in mice were significantly changed, but KO mice had no obvious changes in.8). The immunoblotting experiment showed that the KO mice were not treated with the KO, the expression of the P-STAT3 protein and the mTOR protein, and the isoproteral adrenal gland. After treatment, the expression of FKBP51 protein in WT mice increased and the expression of Collagen I protein and GR protein increased. In addition, the expression of Smad protein in KO mice increased than that of WT mice, indicating that the TGF- beta pathway was closely related to the process. In addition, the expression of mTOR related egg white in KO mice was increased before and after isoproterenol treatment, indicating that mTOR protein was closely related to the process. [conclusion]FKBP51 protein participates in glucocorticoid receptor interaction with glucocorticoid receptor and participates in glucocorticoid signal transduction, through TGF- beta signaling pathway and mTOR signaling pathway to regulate the development of mouse heart and the occurrence of cardiac fibrosis induced by isoproterenol in mice. The purpose is to analyze wild type mice (WT) and Fkbp51 gene knockout (KO). The changes of cardiac mRNA expression pedigree before and after cardiac fibrosis model were constructed with two methods: isoproterenol treatment and left anterior descending branch of heart, respectively, to study the role of Fkbp51 gene in cardiac development and cardiac fibrosis. Methods using the second generation high throughput gene sequencing technology, the treatment and heart of isoproterenol were used. The heart of the two methods of ligation of the left anterior descending branch of the left anterior descending branch was sequenced by the mRNA expression spectrum of the heart of the mice. The sequence data were analyzed with DEGseq, and the differential genes of each group were screened out after limiting the difference conditions. The gene volcano map of the groups was obtained by comparison of the sequence data, and the difference between the groups was further explored. And then make the thermography of each group, then use the online tool DAVID to carry out GO ontology analysis and KEGG pathway analysis, and use Genecards to annotate the gene. Results (1) the absence of Fkbp51 mainly leads to the synthesis and degradation of ribosome related to the ribosome, and the development of the heart can be caused by the functional changes of the specific nucleose body. (2) After the treatment of proterenol, the WT mice showed fibrosis, while the KO mice had no obvious fibrosis.WT mice after the treatment of the PI3K-Akt signaling pathway, and the KO mice were obviously ribosomes. (3) after the ligation of the left anterior descending branch, the two groups of WT and KO mice were all fibroid, but KO mice could have a higher mortality rate of.WT mice. After the main influence of enzyme active agent metabolism, KO mice mainly affect enzyme activity and ionic activity, and influence the infection related pathways to some extent. (4) after ISO treatment in group KO, there is a significant change in ribosome, which is consistent with the changes of Fkbp51 gene knockout. This may be the effect of gene knockout on heart development and ISO treatment. (5) after LAD treatment, the change of WT and KO mice was mainly the change of enzyme activity, but the mortality of KO mice was higher, probably due to the changes of oxidoreductase and susceptibility of related diseases. Conclusion this study was constructed by ligation of the cardiac isoproterenol and left anterior descending branch in the heart of Fkbp51KO and WT mice. The analysis of RNAseq data after cardiac fibrosis shows that the role of Fkbp51 gene in heart development and cardiac fibrosis is a multi gene, multi pathway, multi signal pathway interaction process, which provides a theoretical basis for further study of the molecular mechanism of the role of Fkbp51 gene in heart development and cardiac fibrosis. Lay a theoretical foundation for the possible role of Fkbp51 in heart disease.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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