皮质酮通过Mfn2介导线粒体损伤对鸡胚心脏发育的影响

发布时间：2018-06-02 09:36

本文选题：皮质酮 + 心肌肥大　；参考：《暨南大学》2016年硕士论文

【摘要】：流行病学研究表明,胎儿时期不良的宫内环境会导致成年期高血压、心脏肥大、胰岛素抵抗(Insulin resistance,IR)、2型糖尿病和精神心理障碍等一系列疾病的风险增加。孕妇产前应激状态下,大量分泌的皮质激素(Corticosteroides),能够直接或间接通过胎盘屏障进入宫内影响胚胎发育。近年来更有大量的动物试验及临床研究表明,妊娠期过度的糖皮质激素(Glucocorticoid,GC)暴露与胎儿生长受限、成年期心血管及内分泌代谢等系统性疾病的发生发展密切相关,其中对于心脏的副作用更是包括了短期和长期的影响,因此成为目前研究的关注点之一。然而,关于类固醇皮质激素对胚胎心脏发育的影响及损伤机制尚未深入阐明。同时,考虑到目前用于研究产前皮质类激素暴露对胚胎发育影响过程中胎生动物模型存在胚胎内环境无法接触的局限性。因此,本课题将分别采用鸡胚模型观察应激激素皮质酮(Corticosterone,CORT)对胚胎心脏发育的影响,并结合CORT负荷H9C2细胞模型研究和探讨其内在的分子机理。首先,我们建立CORT负荷诱导心脏发育障碍的鸡胚模型。通过向EDD3(Embryo development day 3)鸡胚气室隔天注射一系列浓度的CORT(0.5、2.5、5、10 nmol/egg),模拟胚胎生长的高皮质类激素暴露环境,继续孵育至EDD14结束。为确定外源性注射的CORT能够进入胚胎体内影响心脏发育,我们通过HPLC-UV方法检测胚胎血浆中CORT的含量,并对胚体及心脏进行称重,统计心脏的相对重量、死亡率等指标。体视镜下观察胚胎心脏的外部形态特征并拍照,经固定、包埋、切片及HE病理染色观察胚胎心脏的内部结构变化。实验结果显示,外源性注射的CORT能够被鸡胚吸收,血浆中CORT水平显著上升,导致胚胎死亡率增加、生长发育迟缓、而心脏相对重量却显著上升,且一定范围内与CORT浓度呈剂量相关性。在此基础上我们选择了CORT 2.5 nmol和5 nmol进行后续实验。形态学结果显示,CORT负荷鸡胚胎的心脏整体体积增大并伴随有心室腔扩大、室壁变薄,室壁心肌细胞密度降低、间距增大等内部形态特征,同时进一步麦胚凝集素(Wheat germ agglutinin,WGA)染色结果显示单个心肌细胞横截面积明显增大。为进一步观察CORT对心脏病理学变化的影响,通过超声心动图(Echocardiography)评价其心脏功能状况,检测结果显示CORT导致胚胎心脏收缩功能下降,表现为射血分数和短轴缩短率显著下降。通过免疫荧光标记技术,我们发现CORT负荷能够抑制心肌细胞的增殖、促进心肌细胞凋亡的发生,同时增加心脏肌球蛋白的表达。RT-PCR检测结果亦发现,经CORT作用后心脏肌动蛋白(Actinαcardiac muscle l,ACTC-1)的mRNA水平显著升高。以上结果提示,cort能够抑制心肌细胞增殖而促进凋亡,刺激细胞向肥大表型转变,造成心脏异常肥大,收缩功能降低。基于心脏组织代谢活跃、能量需求高、线粒体含量丰富等特点,临床上一系列心脏疾病的发生发展均与线粒体代谢功能异常息息相关。相关研究表明,线粒体功能异常被认为是促进心肌病理肥大变化及向心衰转变的主要发病机理之一。为此,本课题进一步研究了cort对鸡胚心肌组织中线粒体功能的影响。向edd3鸡胚气室隔天注射cort(2.5和5nmol)后,孵育至edd14取鸡胚心脏组织,使用线粒体分离试剂盒提取心肌细胞线粒体,采用流式细胞仪检测线粒体膜电位ψm及通过荧光酶标仪实时监测线粒体内ros的生成速率,hplc法测定肥大心肌组织中能量物质atp含量变化。此外,利用试剂盒分别对心肌组织内氧化和抗氧化能力指标、线粒体呼吸链复合物活性进行测定。结果显示,cort负荷能够显著影响鸡胚心肌细胞的线粒体功能,具体表现为ψm下降、ros产生速率增加、atp含量显著下降,脂质过氧化产物mda含量增加而抗氧化酶mnsod活性和抗氧化分子gsh/gssg相对量却显著降低。同时,cort负荷也会导致鸡胚心肌细胞中线粒体呼吸链复合物活性iii、iv、v显著下降。此外,通过透射电镜我们还观察到鸡胚注射cort后,心肌细胞中的线粒体出现聚集呈簇等异常现象。以上结果提示,cort负荷会导致鸡胚心肌细胞中线粒体形态及功能异常。线粒体融合蛋白2(mitofusin2,mfn2)蛋白是一种介导线粒体融合的重要蛋白,参与维持线粒体动力学平衡及结构稳态。近期有研究在啮齿类动物应激模型中发现,应激产生的激素cort能够通过调控mfn2表达而影响脑组织的线粒体结构及功能。因此,我们猜想cort负荷导致胚胎心脏发育紊乱且伴随线粒体功能障碍是否涉及mfn2的表达异常。利用westernblotting检测鸡胚心肌组织mfn2蛋白表达变化,结果表明,cort负荷组鸡胚心肌组织中mfn2蛋白表达水平较对照组明显上调。为了进一步验证cort引起心肌细胞肥大确实是与mfn2介导的线粒体功能缺陷有关,本课题采用cort(200μm,48h)负荷大鼠胚胎心肌细胞h9c2建立体外模型,实验分为control、cort以及cort+gr拮抗剂(ru486)三组,观察cort对h9c2细胞形态的影响,mfn2蛋白水平以及线粒体膜电位的变化,同时通过westernblotting检测细胞水平凋亡相关蛋白的表达情况。最后在单独转染过表达mfn2蛋白情况下,检测其对心肌细胞结构及凋亡的影响,进一步验证cort导致心脏肥大发育异常是通过调控mfn2而实现的。结果显示,h9c2细胞经cort作用后,出现与鸡胚实验相同的结果,cort能够导致心肌细胞体积变大,细胞水平mfn2蛋白表达上调,线粒体膜电位ψm降低以及bcl-2蛋白家族Bax/Bcl-2相对水平增加,心肌细胞凋亡增加。在提前给予GR拮抗剂(Ru486,10μM)基础上再经CORT作用,上述变化均可得到相应的改善,提示CORT可能通过GR介导以上心肌细胞结构及凋亡的改变。同时在转染过表达Mfn2后,凋亡蛋白Bax/Bcl-2显著增加,活化的Caspase3蛋白表达增多以及心肌细胞肥大基因SKA的mRNA水平上升,说明CORT可通过调控Mfn2表达进而损伤线粒体、促进细胞凋亡,致使心室壁细胞数目减少,最终引起心脏发育损伤、异常肥大。综上,CORT通过调控Mfn2介导心肌线粒体功能障碍,导致心肌能量代谢异常、ROS产生过多并且促进心肌细胞凋亡,最终引发心脏发育异常肥大,从而为防治此类相关心脏疾患提供新的治疗靶点和理论依据。
[Abstract]:Epidemiological studies have shown that the undesirable intrauterine environment in the fetal period can lead to adult hypertension, cardiac hypertrophy, insulin resistance (Insulin resistance, IR), the increased risk of a series of diseases such as type 2 diabetes and mental disorder. In pregnant women, a large amount of corticosteroids (Corticosteroides) can be produced directly or between antenatal stressful state. In recent years, a large number of animal experiments and clinical studies have shown that excessive Glucocorticoid (GC) exposure in pregnancy is closely related to the development of fetal growth restriction, cardiovascular and endocrine metabolism in adulthood, including the side of the heart. However, the effect of steroid corticosteroids on fetal heart development and the mechanism of injury have not yet been clarified. Meanwhile, the existence of a fetal animal model in the study of the effects of prenatal corticosteroid exposure on embryonic development is considered. Therefore, we will use the chicken embryo model to observe the effect of Corticosterone (CORT) on the development of fetal heart, and to study and explore the intrinsic molecular mechanism of the CORT load H9C2 cell model. First, we establish the chicken embryo with CORT load induced cardiac dysplasia. Model. By injecting a series of concentrations of CORT (0.5,2.5,5,10 nmol/egg) into the EDD3 (Embryo development day 3) chicken embryo gas chamber every other day to simulate the exposure environment of the high corticosteroid growth of the embryo and continue to incubate to the end of EDD14. In order to determine that the exogenous CORT can enter the fetal body and affect the heart development, we examine the HPLC-UV method. The content of CORT in the plasma of the embryo was measured, and the embryo body and heart were weighed, the relative weight of the heart, the mortality and so on. The external morphological features of the embryonic heart were observed under the body view mirror and photographed. The changes of the internal structure of the embryonic heart were observed by fixed, embedded, sliced and HE pathological staining. The experimental results showed that the exogenous CORT could be injected. The level of CORT in plasma increased significantly, resulting in increased embryo mortality and growth retardation, while the relative weight of the heart increased significantly, and a dose correlation with the concentration of CORT in a certain range. On this basis we chose CORT 2.5 nmol and 5 nmol for follow-up experiments. Morphological results showed that CORT load chicken embryos The overall volume of the heart increased with the enlargement of the ventricular chamber, the thinner wall of the ventricle, the decrease of the cell density and the increase of the space between the ventricular wall, and the further staining of Wheat germ agglutinin (WGA) showed that the cross sectional area of the single cardiac myocytes increased clearly. To further observe the pathological changes of the heart in CORT The results of echocardiography (Echocardiography) were used to evaluate the cardiac function status. The results showed that CORT resulted in a decrease in the systolic function of the embryonic heart, which showed a significant decrease in the ejection fraction and short axis shortening. We found that the CORT load could inhibit the proliferation of cardiac myocytes and promote the apoptosis of cardiac myocytes by immunofluorescent labeling technique. At the same time, the expression of cardiac myosin expression.RT-PCR detection results also found that the mRNA level of Actin alpha cardiac muscle L (ACTC-1) was significantly increased after CORT action. These results suggest that CORT can inhibit the proliferation of cardiac myocytes and promote apoptosis, stimulate cells to change to the hypertrophic phenotype and cause abnormal cardiac hypertrophy. The contractile function is reduced. Based on the active metabolism of the heart, the high energy demand and the abundant mitochondria, the development of a series of heart diseases is closely related to the abnormal mitochondrial metabolic function. The related research shows that the dysfunction of mitochondria is considered to be the main factor to promote the changes of myocardial pathology and to the heart failure. The effect of CORT on mitochondrial function in the myocardium of chicken embryo was further studied. After CORT (2.5 and 5nmol) were injected into the edd3 chicken embryo gas chamber every other day, the heart tissue of chicken embryo was incubated in edd14, the mitochondrial cell line was extracted by the mitochondrial separation kit, and the mitochondrial membrane potential was detected by flow cytometry. M and the real-time monitoring of the formation rate of ROS in mitochondria through a fluorescent enzyme labeling instrument. The change of energy substance ATP content in the hypertrophic myocardium was measured by HPLC method. In addition, the activity of mitochondrial respiratory chain complex was determined by the reagent box, and the activity of mitochondrial respiratory chain complex was determined respectively. The results showed that the CORT load could affect the chicken significantly. The mitochondrial function of embryonic cardiac myocytes is characterized by a decrease in M, an increase in the rate of ROS production, a significant decrease in the content of ATP, the increase in the MDA content of the lipid peroxidation product and a significant decrease in the relative amount of the antioxidant enzyme MnSOD activity and the antioxidant molecule gsh/gssg. Meanwhile, the CORT load will also lead to the activity of mitochondrial respiratory chain complex in the chicken embryo cardiac myocytes. III, IV, V decreased significantly. In addition, through transmission electron microscopy, we also observed that after the injection of CORT, the mitochondria in the cardiac myocytes were clustered and presented clusters of abnormal phenomena. The results suggested that the CORT load could lead to the abnormal mitochondrial morphology and function in the chicken embryo cardiac myocytes. The mitochondrial fusion protein 2 (mitofusin2, Mfn2) protein is a mediating wire. An important protein of granular fusion is involved in maintaining mitochondrial dynamic balance and structural homeostasis. In the recent study in the rodent stress model, stress induced hormone CORT can affect the mitochondrial structure and function of the brain by regulating Mfn2 expression. Therefore, we suspect that the CORT load leads to the disorder of the embryonic heart development and is associated with the disorder of the embryonic heart. The dysfunction of mitochondrial dysfunction involved the abnormal expression of Mfn2. The expression of Mfn2 protein in the myocardium of chicken embryo was detected by westernblotting. The results showed that the expression level of Mfn2 protein in the myocardium of chicken embryo of CORT load group was significantly higher than that of the control group. In order to further verify that CORT induced cardiomyocyte hypertrophy was indeed a line granule mediated by Mfn2. In this study, CORT (200 m, 48h) was used to establish an in vitro model of H9c2 in rat embryonic cardiomyocytes. The experiment was divided into three groups: control, CORT and cort+gr antagonist (RU486). The effect of CORT on the morphology of H9c2 cells, the level of Mfn2 protein and the change of the mitochondrial membrane potential were observed, and the cell water was detected by westernblotting. The expression of flat apoptotic related proteins. Finally, the effects of Mfn2 protein on the structure and apoptosis of cardiac myocytes were detected. The results showed that the abnormal development of cardiac hypertrophy induced by CORT was realized by regulating Mfn2. The results showed that after the action of CORT, the H9c2 cells had the same result as the chicken embryo experiment, CORT It can increase the volume of cardiac myocytes, increase the expression of Mfn2 protein, decrease the mitochondrial membrane potential m and increase the relative level of Bcl-2 protein family Bax/Bcl-2, increase the apoptosis of cardiac myocytes. The changes of the previous changes can be improved accordingly on the basis of GR antagonist (Ru486,10 mu M) in advance, suggesting that CORT may pass through. GR mediated the changes in the structure and apoptosis of the cardiomyocytes. At the same time, after transfection of Mfn2, the apoptotic protein Bax/Bcl-2 increased significantly, the expression of activated Caspase3 protein increased and the mRNA level of cardiac hypertrophy gene SKA increased. It indicated that CORT could damage mitochondria and promote apoptosis by regulating Mfn2 expression, resulting in ventricular wall. The decrease in number of cells leads to cardiac damage and hypertrophy. To sum up, CORT can induce myocardial mitochondrial dysfunction by regulating Mfn2, leading to abnormal cardiac energy metabolism, excessive ROS production and promoting cardiac cell apoptosis, and eventually leading to abnormal hypertrophy of heart development, which provides new therapeutic targets for prevention and control of such related heart diseases. Point and theoretical basis.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965

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