【作者】 历志；

【导师】 易定华； 韩骅； 秦鸿雁；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 心肌梗死随着其发病率和致死率的逐年攀升，已成为严重威胁国民身体健康的第一杀手。急性期心肌梗死主要的死亡原因是急性心衰竭，随着医疗水平的进步，内科药物溶栓、介入支架和外科的搭桥手术使急性期心梗发展成急性心衰进而导致死亡的几率大幅度下降。但随着病情的进一步发展转为慢性期。慢性期主要以心脏纤维化重塑为主，如果纤维重塑过度会引发慢性心衰导致死亡。然而，到目前为止预防和治疗心脏纤维化重塑过度尚无好的方法。有研究表明，巨噬细胞极化在心梗后纤维化重塑全程具有重要的调控作用。那么是否可以通过调控巨噬细胞极化实现对胶原纤维降解和生成的良性调控，进而成为预防和治疗心梗后心室恶性纤维化重塑的新途径呢？目前，许多信号通路参与巨噬细胞的极化。RBP-JК介导的经典Notch信号通路是进化上十分保守的信号通路，对细胞的生长、发育、分化、凋亡及组织重塑等过程起重要的调控作用。我们实验室王耀春博士2010年在实体瘤的研究中首次发现Notch信号与巨噬细胞的极化密切相关，阻断RBP-JК介导的Notch信号可以促进巨噬细胞向M2型极化，而同一年熊思东教授组的研究报道在系统性红斑狼疮中，Notch1通过调控巨噬细胞向M2b的极化而参与了其病理进程。2012年，曹雪涛院士研究组发现，用γ分泌酶抑制剂阻断Notch信号通路会促进TLR诱导的炎症反应，并促进巨噬细胞向M1型极化，同年《Nature Immunol》上发表了Notch信号通路通过上调IRF8促进M1型极化的文章。这些研究均说明Notch信号通路对巨噬细胞的极化起重要的调控作用。然而，RBP-JК介导的Notch信号通路在巨噬细胞参与组织损伤后重塑过程中是否也具有关键的调控作用尚无报道，是否能够通过调节巨噬细胞极化来调节心梗后心室重塑也尚不明确。基于上述问题，本实验首次利用在巨噬细胞中特异剔除Notch信号的小鼠对巨噬细胞在组织损伤重塑过程中是否起作用进行了探索，同时对其在心梗后重塑过程中的调控作用及其机制进行了深入分析，这些研究为寻找治疗和预防心梗后恶性纤维化重塑的新策略提供了理论依据，具有潜在的临床应用价值。目的1．探索RBP-JК介导的Notch信号通路在巨噬细胞参与组织损伤后重塑过程中的作用。2．观察心梗后阻断巨噬细胞中的Notch信号通路对心梗区域及心脏内、外单核/巨噬细胞的影响。3．探寻Notch信号通路调控巨噬细胞参与心梗重塑过程中的分子机制。方法1.构建巨噬细胞中剔除RBP-JК小鼠，并依据实验需要分为实验组（RBP-JК剔除小鼠）和对照组（RBP-JК野生小鼠或杂合小鼠）。2.对12周左右小鼠实验组（10只）和对照组（16只）进行高氏心梗模型的构建，4周之后利用小动物超声对两组小鼠的心脏功能及左室体重比进行比较分析。3.分离心梗后小鼠心脏和肺脏，称重后对两组小鼠心脏体重比和肺脏体重比进行比较分析。4.通过心梗小鼠心脏的大体图片及石蜡切片的Masson染色对两组小鼠心梗面积和纤维化重塑程度进行比较分析。5.对8周左右小鼠实验组（12只）和对照组（12只）建立四氯化碳诱导的组织损伤模型，10周之后分离肝脏、脾脏、肺脏、肾脏及心脏，通过大体图片、组织切片的Masson染色和天狼星红染色对组织损伤程度和纤维化重塑的程度进行比较分析。6.通过心脏石蜡切片的免疫荧光染色对两组小鼠心梗区巨噬细胞数量及类型、细胞凋亡及新生血管数量进行比较分析。7.通过ELISA对两组小鼠非梗死区HYP含量进行比较分析。8.通过Q-PCR对两组小鼠梗死区炎症因子、抗炎因子及纤维化相关因子转录水平进行比较分析。9.采集心梗后两组小鼠的血液并离心获得上清，通过ELISA对血清中炎症因子（TNF-α，IL-6）及抗炎因子（IL-10）进行比较分析。10.获得心梗后两组小鼠的血液、脾脏和骨髓，通过流式对心梗外组织中的单核/巨噬细胞进行比较分析。11.采用LPS加INF-γ诱导原代培养骨髓源的巨噬细胞向炎症相关M1型巨噬细胞极化，通过Western blot、流式和ELISA对两组小鼠炎症相关因子的表达及相关调控通路NF-κB信号通路进行比较分析。结果1．心梗4周后，两组小鼠生存曲线无明显区别。2．心梗4周后，小动物超声显示：敲除小鼠射血分数（EF%）（54.04±7.293）高于对照组（36.03±4.034）；左室短轴收缩率（FS%）(27.62±4.293)高于对照组（17.31±2.180）；敲除小鼠左室舒张期前壁LVAWd(mm)（0.8613±0.07792）、后壁LVPWd(mm)（0.6676±0.01436）及室间隔厚度LVIWd(mm)（3.367±0.2837）薄于对照组（分别为0.6465±0.03634；0.7284±0.06317；4.382±0.1748）；敲除小鼠左室舒张期容积LV Vol d (ul)（47.92±10.31）少于对照组（87.87±8.209）。3．心梗4周后，敲除小鼠左室体重比、心脏体重比及肺脏体重比均低于野生组。4．心梗4周后，心脏大体图片和组织切片MASSON染色均显示心梗后敲除小鼠心脏梗死面积减少；此外，心脏MASSON染色还显示敲除心梗区心肌存活增多，梗死区域及非梗死区域纤维化重塑减轻。5．在四氯化碳诱导的组织损伤模型中，10周后分离小鼠的肝脏、肺脏、脾脏、肾脏及心脏，通过大体图片、Masson染色及天狼猩红染色对两组小鼠进行比较分析发现，敲除小鼠肝脏、肺脏、脾脏、肾脏及心脏损伤轻于对照组，同时各个器官的纤维化重塑程度也轻于对照组。6．心梗4周后，通过免疫荧光染色，在激光共聚焦显微镜下对两组小鼠进行比较分析发现心梗后敲除小鼠梗死区域巨噬细胞减少，主要以炎症相关的巨噬细胞减少为主，抗炎相关的巨噬细胞变化不明显，梗死区域细胞凋亡减少，心梗区域、交接区域和非梗死区域新生血管无明显统计学差异。7．心梗4周后，ELISA检测发现敲除小鼠血中炎症相关因子(TNF-α，IL-6)和抗炎相关因子（IL-10)与对照组相比较变化不明显。8．心梗4周后，大体图片和脾脏体重比显示敲除小鼠脾脏增大不明显。9．心梗4周后，流式细胞仪检测：心梗后敲除小鼠血、骨髓及脾脏中炎症相关（LY6CHighCD11b+）和抗炎相关（LY6CLowCD11b+）的单核细胞减少。此外，敲除小鼠脾脏中单核细胞来源的巨噬细胞（CD11b+F4/80+）减少，且炎症相关的巨噬细胞（F4/80+iNOS+）减少。10．LPS+INF-γ刺激原代培养巨噬细胞2小时后，通过共聚焦显微镜检测：敲除小鼠P65入核减少，IκB降解减少。流式检测：敲除小鼠炎症相关因子TNF-α表达减少。11．LPS+INF-γ刺激原代培养巨噬细胞24小时后，细胞培养液ELISA检测：敲除小鼠巨噬细胞分泌炎症相关因子（TNF-α，IL-6，IL-12）降低，抗炎相关因子(IL-10)升高。共聚焦显微镜检测：敲除小鼠炎症相关因子TNF-α表达减少，P65和P50表达降低，IκB降解降低。蛋白印迹检测：敲除小鼠巨噬细胞中CYLD表达升高，P65和P50表达降低，IκB降解降低。结论1.以两种不同形式的组织慢性损伤模型同时证明了巨噬细胞中RBP-JК介导的Notch信号通路与组织慢性损伤后重塑密切相关。阻断巨噬细胞中的RBP-JК可以减轻组织损伤和纤维化重塑。2.首次发现巨噬细胞中的Notch信号通路与心梗后心脏功能的恢复密切相关。阻断巨噬细胞中的Notch信号通路可以减轻心梗后心脏损伤，抑制纤维化重塑有助于心脏功能的恢复，为改善心梗后心脏功能及治疗和预防心梗后恶性纤维化重塑提供一个新的思路。3.首次发现阻断巨噬细胞中的Notch信号通路可以抑制梗死区域的巨噬细胞向炎症相关M1型极化而对抗炎相关的M2型极化没有影响，并且影响心梗以外其他组织（血液、骨髓及脾脏）中炎症相关单核细胞生成，进而减少炎症相关巨噬细胞的来源，为Notch信号通路在体内调控巨噬细胞极化的机制提供了一个新的思考方向。4.在体外实验中证明了阻断巨噬细胞中的Notch信号可抑制P65及P50的表达，促进CYLD上调抑制IκB降解和P65入核，进而使NF-κB信号通路活化受阻，导致炎症因子分泌减少。同时，我们还发现阻断巨噬细胞中的Notch信号可以促进抗炎相关因子IL-10分泌，提示体外阻断巨噬细胞中的Notch信号可抑制其向M1型极化，促进巨噬细胞向M2型极化。总之，这些研究为寻找治疗和预防心梗后恶性纤维化重塑的新策略提供了理论依据，具有潜在的临床应用价值。更多还原

【Abstract】 Myocardial infarction (MI) has become a serious threat to national healthy withmorbidity and mortality rising year by year. Acute heart failure is the major cause of deathin the acute phase of MI, but with the progress of medical treatment (drug thrombolysis,interventional stent and bypass surgery), the incidence of acute heart failure decreasedsignificantly. With the progression of the disease, it will enter into a chronicphase-ventricular remodeling. Ventricular remodeling following MI is an independent risk factor for many cardiovascular events, which is associated with an inflammatory reaction,followed by scar formation at the site of infarction as well as changes in the non-infarctedmyocardium, including interstitial fibrosis and vascular remodelling. Fibrous tissue thatforms at the site of cardiomyocyte loss preserves structural integrity and is integral to theheart’s recovery, whereas structural remodelling of viable myocardium impairs tissuebehaviour. Therefore, inappropriate ventricular remodeling will seriously affect cardiacfunction, eventually leading to chronic heart failure. Inhibiting inappropriate ventricularremodeling early may be an effective way to prevent or postpone heart failure for patientswith MI. Some studies show that macrophage polarization plays an important role in theregulation of fibrosis remodeling after MI. However, it is unclear that whether appropriatemacrophage polarization could prevente malignant ventricular fibrosis remodeling.Several signaling have been involved in macrophage polarization. TheRBP-JК-mediated Notch signaling is a very evolutionary conserved signaling，which playsan important regulatory role in cell growth, development, differentiation, apoptosis andtissue injury remodeling. In our laboratory, Dr. Wang Yaochun first found that Notchsignaling was closely related to macrophage polarization in mouse tumor model, blockingNotch signaling promoted M2type macrophage polarization. In the same year, Xiong et alreported that Notch1signaling-dependent macrophage M2b polarization might play apivotal role in the pathogenesis of systemic lupus erythematosus.In2012, Cao’s groupfound that blocking the Notch signaling by gamma secretase inhibitors might promoteTLR-induced inflammatory response and promote M1-type macrophages polarization. Inthe same year, one study reported that Notch signaling promotes M1-type polarization byupregulating IRF8that was published by Nature Immunology.Therefore, the Notchsignaling plays an important regulatory role in macrophage polarization. However, it isunclear about the mechanism of RBP-JК-mediated Notch signaling pathway on regulatingmacrophage polarization in fibrosis remodeling after chronic tissue injury is unclear,especially in the case of the MI.In this study, it is the the first time for us to perform comprehensive assessment onthe role of RBP-JК-mediated Notch signaling in regulating macrophage in fibrosis remodeling following chronic tissue injury and possible mechanism of RBP-JК-mediatedNotch signaling on regulating macrophage polarization in fibrosis remodeling followingMI. Our study may provide a novel treatment and prevention strategy on maladaptiveventricular fibrosis remodeling.AIMS1. To investigate whether the RBP-JК-mediated Notch signaling pathway involved inmacrophage regualtion on tissue remodeling following chronic tissue injury.2. To observe whether blocking Notch signaling pathway in macrophage after MI couldinfluence monocyte/macrophage within and outside the heart and infarct area.3. To explore the possible molecular mechanisms of the Notch signaling regulatingmacrophages in ventricular remodeling after MI.METHODS1. To establish conditional RBP-JКknockout mouse in the macrophage. According to theexperiment, the mice could be divided into the experimental group (RBP-JКknockoutmouse) and the control group (Wildtype mouse).2. To establish MI model(Gao Erhe) using12-week-old mice（10experimental mice;16control mice）. Comparative analyses of the heart function between experimental miceand control mice after4weeks by small animal ultrasound instrument.5. Comparative analyses of the heart weight ratio and lung weight ratio betweenexperimental mice and control mice after MI4weeks.6. Comparative analysis of infarct size and collagen content between experimental miceand control mice, which were conducted by light microscope (Masson’s trichromestain) and Stereomicroscope.7. To establish carbon tetrachloride-induced tissue injury model using8-week-old mice（experimental mice,12; control mice,12）. After10weeks,the livers, spleens, lungs,kidneys and hearts were isolated，which were used for comparative analyses of tissueinjury and fibrosis remodeling between experimental mice and control mice by lightmicroscope (Masson’s trichrome stain; Sirius red stain) and Stereomicroscope.8. Comparative analysis of the number and type of macrophage cells, apoptosis and the number of new blood vessels in MI area between experimental mice and control mice,which were conducted by laser scanning confocal microscope (immunofluorescence).9. Comparative analysis of HYP in non-infarcted area between experimental mice andcontrol mice by ELISA.10. Comparative analysis of transcription level of inflammatory cytokines,anti-inflammatory cytokines, and fibrosis-related factor in the infarcted area betweenexperimental mice and control mice by qRT-PCR.11. Comparative analysis of inflammatory cytokines (TNF-α, IL-6) and anti-inflammatorycytokines (IL-10) in serum between experimental mice and control mice by ELISA.12. After MI4weeks the blood, spleens and bone marrow were separated，which wereused for comparative analysis of the number and type of monocytes and macrophagesbetween experimental mice and control mice by flow cytometry.13. To induce primary cultured bone marrow-derived macrophages differentiating toinflammation-related M1–type macrophages under LPS plus INF-γ. Comparativeanalysis of the inflammatory cytokines and their associated NF-κB signaling betweenexperimental mice and control mice by Western blot、qRT-PCR and ELISARESULTS1. There was no significant difference on survival curves between knockout mice (KO)and wild-type mice (WT) after MI4weeks.2. After MI4weeks, the results showed by small animal ultrasound instrument: ejectionfraction (EF%)(54.04±7.293) in knockout group higher than that in the controlgroup (36.03±4.034%); left ventricular fractional shrinkage (FS%)(27.62±4.293)in knockout group higher than that in the control mice(17.31±2.180);left ventriculardiastolic anterior wall LVAWd (mm)(0.8613±0.07792), posterior wall LVPWd (mm)(0.6676±0.01436) and septal thickness LVIWd (mm)(3.367±0.2837) in knockoutmice were thinner than those in the control group (respectively,0.6465±0.03634;0.7284±0.06317;4.382±0.1748); left ventricular diastolic volume of the LVVol d (ul)(47.92±10.31) in knockout mice was less than that in the control group(87.87±8.209). 3. The left ventricular mass, and heart weight ratio and lung weight in the knockout micewere less than those in the wild type mice after MI4weeks.4. Masson staining of cardiac tissue sections showed: in heart of knockout mouse，theinfarct size decreased， myocardial viability increased in MI area， and fibrosisdeposition decreased in the infarct zone and non-infarct zone compared with theexperimental group.5. After carbon tetrachloride induced tissue injury10weeks, mouse livers, lungs, spleens,kidneys and hearts were isolated. We found that the injury in livers, lungs, spleens,kidneys and hearts in knockout mouse was slighter than that in the control group, whilethe extent of fibrosis remodeling of various organs was also slighter than that in thecontrol group by gross pictures, Masson Stain and Picro-Sirius Red Stain.6. After MI4weeks, the result showed that the number of macrophages andinflammation-related macrophages decreased in the infarct area of knockout micecompared with the control group by immunofluorescence staining and laser scanningconfocal microscope, while that there was no statistically significant difference betweenthe two groups in the number of anti-inflammatory-related macrophages. Tunel stainingshowed that cell apoptosis decreased in knockout mouse heart compared with theexperimental group. Immunofluorescence staining of cardiac tissue sections showedthat no statistically significant difference was observed between the two groups inneovascularization in infarction area, the junction of regional and non-infarcted area.7. After MI4weeks, no significant difference on the expression of inflammatorycytokines (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) in serum wasobserved between the two groups.8. After MI4weeks, in knockout mouse, the inflammatory response of spleens were notsignificantly increased through gross pictures and spleen weight ratio compared withthe experimental group. 9. Flow cytometry data showed that: in knockout mouse inflammation related (LY6CHighCD11b+) and anti-inflammatory related (LY6CLowCD11b+) monocytes in blood, bonemarrow and spleen decreased comparing with the experimental group after MI. Inaddition, in knockout mouse, monocyte-derived macrophages (CD11b+F4/80+) andinflammation-associated macrophages (F4/80+iNOS+) in spleen were less than thosein the experimental group.10. Confocal microscopy data showed in the primary cultured macrophage isolated fromknockout mouse the translocation of p65into the nucleus was inhibited and degradationof IκB was reduced compared with the control group after LPS plus INF-γ stimulationfor2hours. As while Flow cytometry data showed the expression ofinflammation-related factor TNF-α in macrophage was decreased in the knockoutmouse compared with the control group.11. Cell supernatant was collected from cultured macrophage and then performed ELISAassay. The results showed secretion of inflammation-related factor (TNF-α, IL-6, IL-12)decreased and anti-inflammatory-related factor (IL-10) increased in the knockout mousecompared with the control group after LPS plus INF-γ stimulation24hours:,. The dataof Immunofluorescence detection showed the expression of inflammation-related factorTNF-α and P65and P50were decreased and degradation of IκB was decreased in themacrophage of knockout mouse compared with the control group. The data of Westernblot showed in the knockout mouse macrophage, the expression of CYLD wasincreased, whereas the expression of P65and P50was decreased and the degradation ofIκB was decreased compared with the control group after LPS plus INF-γ stimulation24hours.CONCLUSIONS1. Using two different models of the mouse chronic injury, such as MI and carbontetrachloride induced organization injury, we verified RBP-JК–dependent Notch signaling in macrophages was closely related to tissue remodeling. To block RBP-JКinmacrophages can reduce chronic injury and suppress fibrosis remodeling.2. It was the first time that we found Notch signaling in macrophages was closelyassociated with the recovery of heart function after MI. It may provide a novel strategyto prevent or treat maladaptive ventricular fibrosis by blocking Notch signalingpathway in macrophages that can reduce cardiac injury and suppress fibrosisrestoration after MI and improve cardiac function.3. Analyzing the macrophage phenotype in the infarct area, we first found that blockingthe Notch signaling in macrophages could reduce the number of M1macrophages butnot affect the number of M2macrophages. Also it can affect the generation ofinflammation-related monocytes out of the MI area, such as blood, bone marrow andspleen. Furthermor it can reduce the number of inflammatory related macrophage.These studies may update the mechanism of the Notch signaling pathway regulationon macrophage polarization.4. Analyzing primary cultured macrophages, we demonstrated that the blockade of theNotch signaling in macrophages might inhibit the expression of P65and the P50andpromote upregulation of CYLD that can prohibit NF-κB signaling pathway bypreventing the translocation of p65into the nucleus and IκB degradation, and finallyreduce the secretion of inflammatory cytokines. Meanwhile, we also found thatblocking the Notch signaling in macrophages could promote the secretion ofanti-inflammatory-related cytokine IL-10. These data suggested blocking Notchsignaling in macrophages in vitro could inhibite M1-type macrophage polarization andpromote M2-type macrophage polarization.Take together, these studies may provide new therapy and prevention strategy onmaladaptive ventricular fibrosis remodeling.更多还原