药物抑制黏着斑激酶改善心肌梗死后纤维化的研究及心脏组织透明化方法初探

发布时间：2018-05-01 23:05

本文选题：黏着斑激酶 + 心脏成纤维细胞　；参考：《北京协和医学院》2015年博士论文

【摘要】：心肌梗死(Myocardial infarction, MI)是临床上常见的极危重症,梗死后的心肌纤维化反应在一定程度上影响着疾病的预后及转归。尽管目前存在许多理论试图解释心肌纤维化的形成机制及其中所涉及的具体信号通路,例如心肌张力／压力的改变,转化生长因子(Transforming growth factor-β, TGF-β)信号通路的激活,以及成纤维细胞向肌成纤维细胞的不当激活等,但是总体而言,心肌纤维化的深层机制仍未完全明了,尚需进一步研究以阐明。结合本课题组以及近年来国内外的科学研究,黏着斑激酶(Focal adhesion kinase, FAK)被认为在纤维化疾病(如皮肤纤维化、肺纤维化与肝纤维化等)中发挥着重要的作用,一方面可以通过介导炎症通路参与纤维化改变,另一方面,FAK可以直接介导成纤维细胞的分化。总而言之,FAK可能作为纤维化疾病的治疗新靶点。但是在缺血缺氧性心脏病,如心肌梗死等病变中,FAK的作用与机制鲜有报道。基于我们前期对FAK在心房颤动心肌纤维化中作用的研究,通过蛋白质组学筛选确定FAK参与心肌纤维化的进程,并介导外源性TGF-β诱导的心脏成纤维细胞分化。在此,我们拟进一步探究：一,心脏成纤维细胞在缺氧条件干预下的分化诱导,FAK是否发挥着重要的介导作用,并且针对其进行靶向干预,可否改善成纤维细胞的分化水平；二,在体内水平,采用构建结扎前降支诱导的小鼠心肌梗死模型,采取药物靶向干预FAK的方法,证实其在梗死后的心肌纤维化中的作用效果。与此同时,结合新近研究发表的被动透明技术(passive clarity technique, PACT),我们定位于心脏组织进行了在检测方法上的探索和改进。在充分保证组织原有结构的前提下,优化心脏组织的透明技术,并通过荧光染色的方法,实现FAK及纤维化相关的胶原蛋白在空间立体结构上的定位表达,并进一步可以推广应用于其它病理状态下的检测。综合以上所述,可以较为系统地解释FAK在心肌梗死(缺血缺氧环境下)心肌纤维化中的作用与机制,为心肌纤维化的治疗提供新的思路和方法,并为心肌纤维化更为精确的检测提供崭新实用的技术支持。主要的研究结果如下：第一部分：黏着斑激酶调控缺氧刺激诱导的心脏成纤维细胞分化目的：研究黏着斑激酶对缺氧刺激诱导的心脏成纤维细胞分化的调控。方法：选择C57BL/6乳鼠,提取原代心脏成纤维细胞并培养,传代至第3代及70%左右细胞融合,以缺氧无血清条件刺激24小时,加用或不用FAK抑制剂——PP2进行干预,分别设为对照组,实验组及干预组。时间终点时,观察细胞形态变化,Western Blot检测FAK活性抑制及下游蛋白AKT信号通路活性改变的情况,免疫荧光检测成纤维细胞分化相关蛋白的表达,Real-time PCR检测FAK及成纤维细胞分化相关基因的表达。结果：原代心脏成纤维细胞在缺氧无血清的条件刺激下,可被诱导分化且FAK活性增高,给予PP2药物干预后,Western Blot验证FAK活性被抑制(p0.05),且Real-time PCR检测FAK、α-SMA及Ⅰ型胶原基因表达水平,实验组明显高于对照组,而药物干预后,FAK (p0.001)、α-SMA (p0.05)及Ⅰ型胶原(p0.05)的基因表达水平明显下降。免疫荧光检测α-SMA及vimentin蛋白表达水平亦呈现药物干预可以使其表达被下调,FAK下游的AKT信号通路参与这一调控过程。结论：缺氧无血清干预可以诱导心脏成纤维细胞分化,给予FAK抑制剂PP2干预后,细胞分化水平可以被明显抑制,AKT信号通路涉及其中。第二部分：黏着斑激酶调控小鼠心肌梗死后心室纤维化目的：研究黏着斑激酶调控小鼠心肌梗死后心室纤维化。方法：选择8-10周龄成年C57BL/6小鼠,构建冠状动脉前降支永久结扎的小鼠心肌梗死模型及未行结扎的假手术模型,模型构建成功的30只小鼠随机分为假手术组,心肌梗死组及药物干预组。其中药物干预组于模型构建成功一周后给予PF-562,271 (15 mg/kg)灌胃处理,持续三周。监测小鼠术后生存状态,并于时间终点时,采用心脏超声检测小鼠心脏功能,心脏组织病理切片以Masson三色及天狼猩红染色检测胶原水平,免疫组化检测FAK及Ⅰ型胶原蛋白表达,Western Blot检测FAK蛋白表达水平,及mTOR, ERK1/2, AKT, P70S6K等FAK信号通路相关蛋白的活性及表达水平。结果：模型构建成功后,心肌梗死组小鼠共有4只分别死于心脏破裂或心功能衰竭,其余两组无死亡。心脏超声功能检测心肌梗死组及药物干预组的小鼠左心室前壁,左心室内径及左心室容积均较假手术组发生明显改变,但比较心脏功能在前述两组间无统计学差异。组织取材后测量心脏、肺脏质量,及心重/体重,模型组均较假手术组增加。Western Blot检测FAK蛋白表达水平因心梗模型构建而明显增加,但药物干预可以明显使之下调。免疫组化检测同样支持这一结果,而且显示Ⅰ型胶原蛋白表达亦与之趋势一致。Masson三色及天狼猩红染色检测胶原水平,在心肌梗死组的梗死周边区明显增加,但药物干预后胶原蛋白表达明显减少。Western Blot检测mTOR, ERK1/2, AKT, P70S6K等FAK信号通路相关蛋白表达水平,因心肌梗死而上调,药物干预可以使之明显下调。结论：在为期4周的心肌梗死状态下,FAK的活性表达增加,并且心脏功能恶化,心室纤维化水平增加。给予药物干预后,FAK活性被抑制,心室纤维化得到明显的改善,并且FAK下游相关蛋白表达水平均发生相应的改变。这些结果说明抑制FAK活性可能为纤维化疾病提供一个有效的治疗方案。第三部分：小鼠心脏组织透明化方法初探目的：初步探索心脏组织透明化的方法及其在心肌纤维化检测中的应用。方法：选择8-10周龄成年C57BL/6小鼠,经下腔静脉灌注PBS flush液清洗组织后,获取心脏并使用4%多聚甲醛固定,垂直于心脏长轴方向,将组织切为1毫米厚度的切片,然后经不同浓度配比的丙烯酰胺+多聚甲醛(分别设置为PBS组,A2P0组,A4P0组,A4P4组,AOP4组)与0.25%VA-044溶液浸泡后制备为组织-凝胶复合物。经组织脱氧后,给予不同浓度的SDS溶液(分别设置PBS组,4%,8%,12%,16%,20%等浓度组)长时间漂洗,可见心脏组织逐渐透明化的程度,期间同时检测组织重量变化,漂洗后总蛋白损失量,并对各样本进行拍照记录及对比。给予心肌纤维化相关蛋白磷酸化黏着斑激酶(p-FAK)及Ⅰ型胶原蛋白(Col-I)抗体孵育后,以激光共聚焦显微镜下检测蛋白表达水平,并可构建3D立体影像。结果：1毫米左右厚度的心脏组织切片在A4P0组条件浸泡下,可形成合适的组织-凝胶单体,而经过8%或16%浓度的SDS溶液漂洗72小时后,可呈现肉眼可见的组织透明化。心脏组织切片经漂洗后,组织重量均有一定程度的增加,但在8%SDS组拥有更少的蛋白损失,且该组样本经抗体孵育后可充分显示心肌组织内p-FAK, Col-I蛋白的空间表达。结论：A4P0结合8%SDS漂洗处理72小时后的1毫米厚度心脏组织切片,可呈现明显透明化,且透明化后的心脏组织切片应用免疫荧光的方法,可用作检测组织原位空间内的蛋白表达水平。
[Abstract]:Myocardial infarction (MI) is a very common critical critically ill. The myocardial fibrosis reaction after infarction affects the prognosis and prognosis of the disease to a certain extent. Although there are many theories trying to explain the formation mechanism of myocardial fibrosis and the specific signal pathways involved, such as myocardial tension / pressure Changes in the activation of the Transforming growth factor- beta (TGF- beta) signaling pathway and the undue activation of fibroblasts to myofibroblasts, but in general, the deep mechanism of myocardial fibrosis is still not fully understood. Further research is needed to elucidate. In study, Focal adhesion kinase (FAK) is considered to play an important role in fibrotic diseases (such as skin fibrosis, pulmonary fibrosis and liver fibrosis). On the one hand, it can mediate the inflammatory pathway to participate in fibrosis, and on the other hand, FAK can directly mediate the differentiation of fibroblasts. In a word, FAK can be used. It can be used as a new target for the treatment of fibrotic diseases. However, there are few reports on the role and mechanism of FAK in ischemic and anoxic heart disease, such as myocardial infarction. Based on our earlier study of the role of FAK in atrial fibrillation, the process of FAK involvement in myocardial fibrosis is identified by proteomics, and exogenous TG is mediated. F- beta induced cardiac fibroblasts differentiation. Here, we intend to further explore: first, the differentiation induction of cardiac fibroblasts under the intervention of hypoxia, whether FAK plays an important mediating role, and targeted intervention to improve the differentiation level of fibroblasts; two, in the body level, the establishment of ligature The model of the mouse myocardial infarction induced by the anterior descending branch, using the method of drug targeting to intervene FAK, confirms its effect in the myocardial fibrosis after the infarction. At the same time, combined with the newly published passive clarity technique (PACT), we have decided to explore and modify the method of detection in the cardiac tissue. Under the premise of fully guaranteeing the original structure of the organization, the transparent technology of the heart tissue is optimized, and the localization and expression of FAK and fibrosis related collagen in the spatial structure can be realized by the method of fluorescent staining, and can be further applied to the detection of other pathological states. To explain the role and mechanism of FAK in myocardial fibrosis in myocardial infarction (ischemic anoxia), provide new ideas and methods for the treatment of myocardial fibrosis, and provide new and practical technical support for more accurate detection of myocardial fibrosis. The main results are as follows: the regulation of hypoxia stimulation induced by focal adhesion kinase The aim of cardiac fibroblast differentiation is to study the regulation of the differentiation of cardiac fibroblasts induced by hypoxia stimulation. Methods: select C57BL/6 mice, extract the primary cardiac fibroblasts and culture, pass the passage to third and 70% cells to fuse for 24 hours with anoxic serum-free conditions, plus or without FAK suppression. The preparation, PP2, was given to the control group, the experimental group and the intervention group. The morphological changes of the cells were observed at the end of the time, the activity of FAK activity and the activity of the downstream protein AKT signaling pathway were detected by Western Blot, the expression of the related protein of fibroblast differentiation was detected by immunofluorescence, and FAK and fibroblasts were detected by Real-time PCR. The expression of differentiation related genes. Results: the primary cardiac fibroblasts could be induced to differentiate and increase the activity of FAK under the condition of hypoxia and serum free conditions. The prognosis of PP2 drugs was given, and the activity of FAK was inhibited by Western Blot (P0.05), and Real-time PCR detected FAK, alpha -SMA and type I collagen gene expression level, the experimental group was significantly higher than that of the experimental group. The gene expression level of FAK (p0.001), alpha -SMA (P0.05) and type I collagen (P0.05) decreased significantly in the group of drugs, and the expression level of alpha -SMA and vimentin protein expressed by immunofluorescence also showed that drug intervention could reduce its expression and participate in the regulation process of AKT signal through the downstream of FAK. Conclusion: anoxia free serum intervention can be used. Induction of cardiac fibroblast differentiation, FAK inhibitor PP2 dry prognosis, the level of cell differentiation can be significantly inhibited, AKT signaling pathway involved. The second part: adhesion kinase regulation of ventricular fibrosis in mice after myocardial infarction: To study the regulation of ventricular fibrosis after myocardial infarction in mice. Method: select 8-10 In the adult C57BL/6 mice of week age, a model of myocardial infarction and a ligation model were constructed for permanent ligation of the anterior descending branch of the coronary artery. The 30 mice were randomly divided into the sham operation group, the myocardial infarction group and the drug intervention group. The drug intervention group was given PF-562271 (15 mg/kg) perfusion one week after the model construction was successful. Gastric treatment lasted three weeks. The survival status of mice was monitored and the cardiac function of mice was detected by echocardiography at the end of time. The pathological sections of the heart were detected by Masson trichrome and Sirius scarlet staining, FAK and type I collagen were detected by immunohistochemistry, the expression of FAK protein was detected by Western Blot, and mTOR, ERK The activity and expression level of FAK signal pathway related proteins such as 1/2, AKT and P70S6K. Results: after the model construction was successful, 4 of the mice in the myocardial infarction group died of cardiac rupture or heart failure respectively, and the other two groups were not dead. The echocardiography function of the myocardial infarction group and the drug intervention group was tested for the left ventricular anterior wall of the mice and the left ventricular diameter and the diameter of the left ventricle. The left ventricular volume was significantly higher than that in the sham operation group, but there was no statistical difference between the two groups. The heart, lung quality and heart weight / weight were measured after tissue sampling. The model group increased the level of FAK protein in the model group compared with the sham operation group by.Western Blot, but the drug intervention could be increased. Immunohistochemical detection also supported this result, and showed that the expression of type I collagen was also consistent with the trend of.Masson tricolor and Sirius scarlet staining to detect the collagen level, which was significantly increased in the infarct surrounding area of the myocardial infarction group, but the expression of collagen egg white was significantly reduced by.Western Blot after the drug intervention, and mTOR was detected, ERK1/ 2, AKT, P70S6K and so on, the expression level of FAK signal pathway related protein is up regulated by myocardial infarction, and the drug intervention can be reduced obviously. Conclusion: the expression of FAK activity is increased in the state of myocardial infarction for 4 weeks, and the cardiac function is deteriorated, the level of ventricular fibrosis is increased. The activity of FAK is suppressed, the activity of FAK is inhibited and ventricular fibers are inhibited. The results showed that the expression level of FAK downstream related proteins changed correspondingly. These results suggest that inhibition of FAK activity may provide an effective treatment for fibrotic diseases. Third part: preliminary exploration of the method of transparency of cardiac tissue in mice Methods: the use of the detection of muscle fibrosis. Methods: select 8-10 weeks old adult C57BL/6 mice, after the inferior vena cava perfusion PBS flush liquid cleaning tissue, obtain the heart and use 4% polyformaldehyde fixed, vertical to the long axis of the heart, cut the tissue to 1 millimeter thickness section, and then the same concentration of acrylamide + polyformaldehyde (respectively set up) PBS group, group A2P0, group A4P0, group A4P4, AOP4 group and 0.25%VA-044 solution were soaked with 0.25%VA-044 solution to prepare tissue gel complex. After tissue deoxidation, different concentrations of SDS solution (set PBS group, 4%, 8%, 12%, 16%, 20% concentration groups) were rinsed for a long time, and the degree of gradual transparency of the heart tissue was seen, and the tissue weight change was detected at the same time. The total protein loss after rinsing was recorded and compared. The protein expression level of p-FAK and type I collagen (Col-I) antibody was incubated with myocardial fibrosis related protein phosphorylated adhesion kinase (p-FAK) and type I collagen (type I collagen). The protein expression level was detected by laser confocal microscope, and the 3D stereoscopic image could be constructed. Under the condition of A4P0 group, the dirty tissue section can form a suitable tissue gel monomer, and after rinsing for 72 hours after 8% or 16% concentration of SDS solution, the tissue transparency can be seen. After rinsing the tissue section, the tissue weight increases to a certain extent, but there is less protein loss in the 8%SDS group, and the group sample is less than that of the group. This antibody can fully display the spatial expression of p-FAK and Col-I protein in the myocardium. Conclusion: the 1 mm thickness of heart tissue section of A4P0 combined with 8%SDS rinsing for 72 hours can be clearly transparent, and the transparent heart tissue section can be used as a method of immunofluorescence to detect the eggs in the tissue in situ. White expression level.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.22

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