本文選題：黏著斑激酶 + 心臟成纖維細胞��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：心肌梗死(Myocardial infarction, MI)是臨床上常見的極危重癥,梗死后的心肌纖維化反應(yīng)在一定程度上影響著疾病的預(yù)后及轉(zhuǎn)歸。盡管目前存在許多理論試圖解釋心肌纖維化的形成機制及其中所涉及的具體信號通路,例如心肌張力／壓力的改變,轉(zhuǎn)化生長因子(Transforming growth factor-β, TGF-β)信號通路的激活,以及成纖維細胞向肌成纖維細胞的不當激活等,但是總體而言,心肌纖維化的深層機制仍未完全明了,尚需進一步研究以闡明。結(jié)合本課題組以及近年來國內(nèi)外的科學(xué)研究,黏著斑激酶(Focal adhesion kinase, FAK)被認為在纖維化疾病(如皮膚纖維化、肺纖維化與肝纖維化等)中發(fā)揮著重要的作用,一方面可以通過介導(dǎo)炎癥通路參與纖維化改變,另一方面,FAK可以直接介導(dǎo)成纖維細胞的分化�？偠灾�,FAK可能作為纖維化疾病的治療新靶點。但是在缺血缺氧性心臟病,如心肌梗死等病變中,FAK的作用與機制鮮有報道�；谖覀兦捌趯AK在心房顫動心肌纖維化中作用的研究,通過蛋白質(zhì)組學(xué)篩選確定FAK參與心肌纖維化的進程,并介導(dǎo)外源性TGF-β誘導(dǎo)的心臟成纖維細胞分化。在此,我們擬進一步探究：一,心臟成纖維細胞在缺氧條件干預(yù)下的分化誘導(dǎo),FAK是否發(fā)揮著重要的介導(dǎo)作用,并且針對其進行靶向干預(yù),可否改善成纖維細胞的分化水平；二,在體內(nèi)水平,采用構(gòu)建結(jié)扎前降支誘導(dǎo)的小鼠心肌梗死模型,采取藥物靶向干預(yù)FAK的方法,證實其在梗死后的心肌纖維化中的作用效果。與此同時,結(jié)合新近研究發(fā)表的被動透明技術(shù)(passive clarity technique, PACT),我們定位于心臟組織進行了在檢測方法上的探索和改進。在充分保證組織原有結(jié)構(gòu)的前提下,優(yōu)化心臟組織的透明技術(shù),并通過熒光染色的方法,實現(xiàn)FAK及纖維化相關(guān)的膠原蛋白在空間立體結(jié)構(gòu)上的定位表達,并進一步可以推廣應(yīng)用于其它病理狀態(tài)下的檢測。綜合以上所述,可以較為系統(tǒng)地解釋FAK在心肌梗死(缺血缺氧環(huán)境下)心肌纖維化中的作用與機制,為心肌纖維化的治療提供新的思路和方法,并為心肌纖維化更為精確的檢測提供嶄新實用的技術(shù)支持。主要的研究結(jié)果如下：第一部分：黏著斑激酶調(diào)控缺氧刺激誘導(dǎo)的心臟成纖維細胞分化目的：研究黏著斑激酶對缺氧刺激誘導(dǎo)的心臟成纖維細胞分化的調(diào)控。方法：選擇C57BL/6乳鼠,提取原代心臟成纖維細胞并培養(yǎng),傳代至第3代及70%左右細胞融合,以缺氧無血清條件刺激24小時,加用或不用FAK抑制劑——PP2進行干預(yù),分別設(shè)為對照組,實驗組及干預(yù)組。時間終點時,觀察細胞形態(tài)變化,Western Blot檢測FAK活性抑制及下游蛋白AKT信號通路活性改變的情況,免疫熒光檢測成纖維細胞分化相關(guān)蛋白的表達,Real-time PCR檢測FAK及成纖維細胞分化相關(guān)基因的表達。結(jié)果：原代心臟成纖維細胞在缺氧無血清的條件刺激下,可被誘導(dǎo)分化且FAK活性增高,給予PP2藥物干預(yù)后,Western Blot驗證FAK活性被抑制(p0.05),且Real-time PCR檢測FAK、α-SMA及Ⅰ型膠原基因表達水平,實驗組明顯高于對照組,而藥物干預(yù)后,FAK (p0.001)、α-SMA (p0.05)及Ⅰ型膠原(p0.05)的基因表達水平明顯下降。免疫熒光檢測α-SMA及vimentin蛋白表達水平亦呈現(xiàn)藥物干預(yù)可以使其表達被下調(diào),FAK下游的AKT信號通路參與這一調(diào)控過程。結(jié)論：缺氧無血清干預(yù)可以誘導(dǎo)心臟成纖維細胞分化,給予FAK抑制劑PP2干預(yù)后,細胞分化水平可以被明顯抑制,AKT信號通路涉及其中。第二部分：黏著斑激酶調(diào)控小鼠心肌梗死后心室纖維化目的：研究黏著斑激酶調(diào)控小鼠心肌梗死后心室纖維化。方法：選擇8-10周齡成年C57BL/6小鼠,構(gòu)建冠狀動脈前降支永久結(jié)扎的小鼠心肌梗死模型及未行結(jié)扎的假手術(shù)模型,模型構(gòu)建成功的30只小鼠隨機分為假手術(shù)組,心肌梗死組及藥物干預(yù)組。其中藥物干預(yù)組于模型構(gòu)建成功一周后給予PF-562,271 (15 mg/kg)灌胃處理,持續(xù)三周。監(jiān)測小鼠術(shù)后生存狀態(tài),并于時間終點時,采用心臟超聲檢測小鼠心臟功能,心臟組織病理切片以Masson三色及天狼猩紅染色檢測膠原水平,免疫組化檢測FAK及Ⅰ型膠原蛋白表達,Western Blot檢測FAK蛋白表達水平,及mTOR, ERK1/2, AKT, P70S6K等FAK信號通路相關(guān)蛋白的活性及表達水平。結(jié)果：模型構(gòu)建成功后,心肌梗死組小鼠共有4只分別死于心臟破裂或心功能衰竭,其余兩組無死亡。心臟超聲功能檢測心肌梗死組及藥物干預(yù)組的小鼠左心室前壁,左心室內(nèi)徑及左心室容積均較假手術(shù)組發(fā)生明顯改變,但比較心臟功能在前述兩組間無統(tǒng)計學(xué)差異。組織取材后測量心臟、肺臟質(zhì)量,及心重/體重,模型組均較假手術(shù)組增加。Western Blot檢測FAK蛋白表達水平因心梗模型構(gòu)建而明顯增加,但藥物干預(yù)可以明顯使之下調(diào)。免疫組化檢測同樣支持這一結(jié)果,而且顯示Ⅰ型膠原蛋白表達亦與之趨勢一致。Masson三色及天狼猩紅染色檢測膠原水平,在心肌梗死組的梗死周邊區(qū)明顯增加,但藥物干預(yù)后膠原蛋白表達明顯減少。Western Blot檢測mTOR, ERK1/2, AKT, P70S6K等FAK信號通路相關(guān)蛋白表達水平,因心肌梗死而上調(diào),藥物干預(yù)可以使之明顯下調(diào)。結(jié)論：在為期4周的心肌梗死狀態(tài)下,FAK的活性表達增加,并且心臟功能惡化,心室纖維化水平增加。給予藥物干預(yù)后,FAK活性被抑制,心室纖維化得到明顯的改善,并且FAK下游相關(guān)蛋白表達水平均發(fā)生相應(yīng)的改變。這些結(jié)果說明抑制FAK活性可能為纖維化疾病提供一個有效的治療方案。第三部分：小鼠心臟組織透明化方法初探目的：初步探索心臟組織透明化的方法及其在心肌纖維化檢測中的應(yīng)用。方法：選擇8-10周齡成年C57BL/6小鼠,經(jīng)下腔靜脈灌注PBS flush液清洗組織后,獲取心臟并使用4%多聚甲醛固定,垂直于心臟長軸方向,將組織切為1毫米厚度的切片,然后經(jīng)不同濃度配比的丙烯酰胺+多聚甲醛(分別設(shè)置為PBS組,A2P0組,A4P0組,A4P4組,AOP4組)與0.25%VA-044溶液浸泡后制備為組織-凝膠復(fù)合物。經(jīng)組織脫氧后,給予不同濃度的SDS溶液(分別設(shè)置PBS組,4%,8%,12%,16%,20%等濃度組)長時間漂洗,可見心臟組織逐漸透明化的程度,期間同時檢測組織重量變化,漂洗后總蛋白損失量,并對各樣本進行拍照記錄及對比。給予心肌纖維化相關(guān)蛋白磷酸化黏著斑激酶(p-FAK)及Ⅰ型膠原蛋白(Col-I)抗體孵育后,以激光共聚焦顯微鏡下檢測蛋白表達水平,并可構(gòu)建3D立體影像。結(jié)果：1毫米左右厚度的心臟組織切片在A4P0組條件浸泡下,可形成合適的組織-凝膠單體,而經(jīng)過8%或16%濃度的SDS溶液漂洗72小時后,可呈現(xiàn)肉眼可見的組織透明化。心臟組織切片經(jīng)漂洗后,組織重量均有一定程度的增加,但在8%SDS組擁有更少的蛋白損失,且該組樣本經(jīng)抗體孵育后可充分顯示心肌組織內(nèi)p-FAK, Col-I蛋白的空間表達。結(jié)論：A4P0結(jié)合8%SDS漂洗處理72小時后的1毫米厚度心臟組織切片,可呈現(xiàn)明顯透明化,且透明化后的心臟組織切片應(yīng)用免疫熒光的方法,可用作檢測組織原位空間內(nèi)的蛋白表達水平。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R542.22

【參考文獻】

相關(guān)期刊論文 前2條

1 郝嘉;嚴俊;郭紅;肖穎彬;;RNA干擾技術(shù)沉默黏附斑激酶誘導(dǎo)心臟成纖維細胞失巢凋亡[J];中國臨床康復(fù);2006年25期

2 宋凡偉;朱世澤;;黏著斑激酶與纖維化疾病關(guān)系的研究進展[J];中華臨床醫(yī)師雜志(電子版);2011年23期

，

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