發(fā)布時(shí)間：2018-05-30 22:05

  本文選題：FKBP51 + 心臟纖維化　； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文

【摘要】：[目的]心臟纖維化是以心臟間質(zhì)細(xì)胞外基質(zhì)蛋白積累為特點(diǎn),造成心臟收縮和舒張功能不全,是許多心臟病理生理變化的前提條件。糖皮質(zhì)激素(GC)是一類由腎上腺皮質(zhì)分泌的甾體激素,不但可以通過糖皮質(zhì)激素受體(GR)調(diào)節(jié)糖、脂肪和蛋白質(zhì)的生物合成及代謝,而且糖皮質(zhì)激素在心血管系統(tǒng)的平衡中也具有重要作用;作為糖皮質(zhì)激素受體的共伴侶蛋白,FKBP51蛋白參與糖皮質(zhì)激素受體介導(dǎo)的信號傳導(dǎo)過程,因此敲除Fkbp51可能對心臟發(fā)育和心臟纖維化的發(fā)生產(chǎn)生影響。本論文通過建立Fkbp51基因敲除小鼠心臟纖維化模型,探究FKBP51在心臟發(fā)育及心臟纖維化中的作用,為探究心臟發(fā)育和研制抗心臟纖維化藥物提供理論依據(jù)。[方法]利用實(shí)驗(yàn)室已有的Fkbp51基因敲除(KO)小鼠,通過建立心臟纖維化模型,分析Fkbp51基因在心臟纖維化過程中的作用。1)小鼠的心臟纖維化模型建立:將實(shí)驗(yàn)小鼠分為四組(每組五只),分別為皮下種植緩釋泵緩慢釋放生理鹽水的野生型小鼠(WT-control)、釋放生理鹽水的Fkbp51基因敲除小鼠(KO-control)、釋放異丙腎上腺素(ISO)的野生型小鼠(WT-ISO)、釋放異丙腎上腺素的Fkbp51基因敲除小鼠(KO-ISO),持續(xù)三周;2)心臟超聲與心電功能檢測:對以上實(shí)驗(yàn)組中小鼠麻醉后,進(jìn)行超聲與心電功能檢測;3)心臟表觀病理分析:使用電子天平稱量實(shí)驗(yàn)小鼠心臟重量,使用相機(jī)對實(shí)驗(yàn)小鼠心臟進(jìn)行拍照并觀察其大小和形態(tài);對各實(shí)驗(yàn)組小鼠心臟進(jìn)行常規(guī)石蠟包埋并切片,進(jìn)行HE染色和Masson染色,探究WT和KO小鼠心臟組織病理學(xué)變化;4)利用Real-time PCR技術(shù)檢測心臟纖維化相關(guān)基因mRNA水平上的變化;5)利用免疫組化和免疫熒技術(shù)檢測心臟纖維化相關(guān)基因蛋白水平上的變化;6)血清指標(biāo)檢測。對四組小鼠的血清進(jìn)行心臟纖維化相關(guān)炎癥因子和細(xì)胞因子驗(yàn)證;7)利用免疫印跡技術(shù)檢測(1中四組小鼠)心臟組織中纖維化標(biāo)志性蛋白、TGF-β通路相關(guān)蛋白、m-TOR通路相關(guān)蛋白的變化。[結(jié)果]1)通過免疫組織化學(xué)方法鑒定證明成功構(gòu)建了 WT心臟纖維化動物模型,而Fkbp51KO小鼠并無明顯纖維化出現(xiàn)。2)通過超聲與心電檢測,顯示Fkbp51基因敲除后,Fkbp51 KO小鼠主波電壓平均水平顯著高于WT小鼠且心率顯著減慢,表明KO小鼠可能存在房室傳導(dǎo)阻滯,且KO小鼠心室肌厚度大于WT小鼠心室肌厚度,心臟發(fā)育異常。ISO處理后,超聲結(jié)果顯示,WT小鼠的射血分?jǐn)?shù)、舒張末期左室后壁厚度及左室短軸縮短率均降低,心功能顯著降低;而KO小鼠并無顯著性變化,心功能相對穩(wěn)定。心電結(jié)果顯示W(wǎng)T主波明顯變深變高,KO主波變高趨勢不如WT明顯且心率明顯加快,傳導(dǎo)也加快,收縮力增強(qiáng)。3)通過對心臟表觀進(jìn)行觀察發(fā)現(xiàn):ISO處理組中,WT小鼠心臟明顯變大、變重,而Fkbp51 KO心臟無明顯改變。4)心臟病理學(xué)分析發(fā)現(xiàn),ISO處理后,WT小鼠心臟出現(xiàn)明顯肥大,心臟壁變薄,出現(xiàn)大量纖維化;而Fkbp51基因敲除小鼠并未出現(xiàn)纖維化,且心臟壁無明顯改變。5)通過Real-time PCR檢測,發(fā)現(xiàn)ISO處理后Fkbp51基因顯著增加;Collagen Ⅰ和AngⅡ在Fkbp51基因敲除小鼠中的表達(dá)明顯少于野生型小鼠,異丙腎上腺素處理后WT小鼠出現(xiàn)顯著性升高,而KO小鼠無明顯改變;與心臟纖維化Marker相關(guān)的細(xì)胞因子TGF-β以及TGF-βR Ⅰ、TGF-βRⅡ也出現(xiàn)此趨勢。6)免疫熒光實(shí)驗(yàn),對Collagen Ⅰ蛋白進(jìn)行驗(yàn)證,發(fā)現(xiàn)其在Fkbp51基因敲除小鼠心臟中的表達(dá)明顯少于野生型小鼠;免疫組化實(shí)驗(yàn),對α-SMA和TIMP1等纖維化標(biāo)志蛋白進(jìn)行驗(yàn)證,發(fā)現(xiàn)在Fkbp51基因敲除小鼠中的表達(dá)明顯少于野生型小鼠。7)血清指標(biāo)檢驗(yàn)發(fā)現(xiàn),ISO處理后纖維化標(biāo)志分子小鼠成纖維細(xì)胞生長因子、小鼠超氧化物歧化酶、血管緊張素Ⅱ和小鼠L—乳酸脫氫酶、小鼠腫瘤壞死因子α和小鼠γ干擾素出現(xiàn)顯著性改變,而KO小鼠無明顯改變。8)免疫印跡實(shí)驗(yàn)發(fā)現(xiàn):未經(jīng)處理時(shí),KO小鼠α-SMA蛋白、P-STAT3蛋白和mTOR蛋白表達(dá)增加,異丙腎上腺素處理后WT小鼠FKBP51蛋白表達(dá)增加,且CollagenⅠ蛋白和GR蛋白表達(dá)也增加。此外KO小鼠比WT小鼠Smad蛋白表達(dá)增加,說明TGF-β通路與此過程存在密切相關(guān),此外異丙腎上腺素處理前后,KO小鼠mTOR相關(guān)蛋白表達(dá)增加,說明mTOR蛋白與此過程密切相關(guān)。[結(jié)論]FKBP51蛋白通過與糖皮質(zhì)激素受體相互作用,參與糖皮質(zhì)激素信號傳導(dǎo),通過TGF-β信號通路和mTOR信號通路調(diào)控小鼠心臟發(fā)育和由異丙腎上腺素誘導(dǎo)的小鼠心臟纖維化的發(fā)生。目的通過分析野生型小鼠(WT)和Fkbp51基因敲除(KO)小鼠分別使用異丙腎上腺素處理和心臟左前降支結(jié)扎兩種方法構(gòu)建心臟纖維化模型前后心臟mRNA表達(dá)系譜的變化,研究Fkbp51基因在心臟發(fā)育和心臟纖維化過程中的作用。方法利用第二代高通量基因測序技術(shù),對異丙腎上腺素處理和心臟左前降支結(jié)扎兩種方法構(gòu)建的纖維化模型小鼠的心臟進(jìn)行mRNA表達(dá)譜測序,將測序的數(shù)據(jù)用DEGseq進(jìn)行差異分析,限定差異條件后篩選出各組小鼠心臟的差異基因,通過測序數(shù)據(jù)各組間比較得到組間的基因火山圖,進(jìn)一步探索組間差別并做出各組的熱圖,之后利用在線工具DAVID對差異基因進(jìn)行GO本體分析和KEGG通路分析,并利用Genecards對基因進(jìn)行注釋。結(jié)果(1)Fkbp51的缺失主要導(dǎo)致與核糖體相關(guān)的合成與降解,通過特異性的核糖體的功能變化引起心臟的發(fā)育損傷。(2)異丙腎上腺素處理后,WT小鼠呈現(xiàn)出纖維化,而KO小鼠無明顯纖維化。WT小鼠處理后PI3K-Akt信號通路出現(xiàn)明顯改變,而KO小鼠存在明顯改變的是核糖體。(3)左前降支結(jié)扎后,WT和KO小鼠兩組均存在纖維化,但KO小鼠會有比較高的死亡率。WT小鼠處理后主要影響酶活性劑代謝,KO小鼠主要影響酶活性和離子活性,并一定程度上影響感染相關(guān)的通路。(4)KO組ISO處理后,存在顯著性改變的依然是核糖體,與Fkbp51基因敲除后的變化一致,這可能是基因敲除后影響心臟發(fā)育及ISO處理后抗纖維化的原因。(5)LAD處理后,WT和KO小鼠相比,改變的主要是酶的活性變化,但KO小鼠存在較高的死亡率,可能是由于氧化還原酶的變化及相關(guān)疾病的易感性。結(jié)論本研究通過對Fkbp51KO與WT小鼠的心臟異丙腎上腺素和左前降支結(jié)扎兩種方法構(gòu)建心臟纖維化后的RNAseq數(shù)據(jù)進(jìn)行分析,發(fā)現(xiàn)Fkbp51基因在心臟發(fā)育和心臟纖維化中的作用是一個(gè)多基因、多途徑、多信號通路相互作用的過程,為下一步深入研究Fkbp51基因在心臟發(fā)育和心臟纖維化中作用的分子機(jī)制提供了理論基礎(chǔ),進(jìn)一步為Fkbp51在心臟疾病中可能起到的作用做出理論鋪墊。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

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