【作者】 程轶喆；

【导师】 苗俊英；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 博士

【摘要】 研究背景和目的血管内皮细胞（VEC）在血管发育中起重要作用。大量实验结果表明,血管内皮细胞凋亡导致的血管功能异常,将引起多种炎症和退行性疾病的发生。这些重大疾病严重危害人类健康,如何防治这些疾病是目前亟待解决的问题。新血管形成（angiogenesis）在生理过程（如胚胎发育）和病理过程（如肿瘤生长、动脉粥样硬化）中都起到关键作用。新血管形成与内皮细胞凋亡有密切关系,抑制血管内皮细胞凋亡是促进新血管形成的关键因素,但是两者关联的分子机制目前尚未搞清。从化学遗传学的角度,利用小分子化合物可以发现参与血管内皮细胞凋亡和血管形成的新关键因子,因此可为阐明上述科学问题提供实验证据。先前的研究证明去除血清和生长因子可诱导人脐静脉内皮细胞（HUVEC）凋亡,在此条件下,6-氨基-2,3-二氢-3-羟甲基-1,4-苯并噁嗪衍生物（ABO）能提高血管内皮细胞的存活率,因此推断ABO可能是血管内皮细胞凋亡抑制剂和血管形成促进剂。本论文研究目的是从化学遗传学角度,以小分子化合物ABO为工具,研究血管内皮细胞凋亡、血管形成以及两者关联的分子机制。为血管退行性重大疾病的防治提供新线索和靶点。已知细胞内的氧化还原反应（redox）在细胞凋亡和血管形成中起重要作用,参与redox的成分包括:线粒体、活性氧（ROS）、NADPH氧化酶、超氧化物歧化酶（SOD）以及一氧化氮（NO）/内皮型一氧化氮合酶（eNOS）等。因此,本论文首先研究了ABO对redox信号分子的调节机制。随后研究了在ABO的作用下,其它与凋亡和成血管密切相关的分子（如:磷脂酰胆碱特异性磷脂酶C（PC-PLC）、P53、膜整连蛋白β₄、核因子κB（NF-κB）以及H-ras）的变化情况。研究内容1.ABO抑制去除血清和生长因子诱导血管内皮细胞凋亡的作用2.ABO促进血管生成的作用3.ABO抑制血管内皮细胞凋亡和促进血管生成的分子机制研究研究方法1.血管内皮细胞培养:人脐静脉内皮细胞的提取和培养参考Jaffe EA et al的方法[Jaffe EA et al,1973]2.细胞凋亡检测:1)MTT检测细胞存活率2)倒置相差显微镜观察细胞形态变化3)吖啶橙染色结合激光扫描共聚焦显微镜,观察细胞核凝集及片断化4)TUNEL方法,检测细胞凋亡率3.体外血管形成检测:Matrigel方法,参考[Kureishi Y et al,2000]4.体内血管形成检测:鸡胚尿囊膜（CAM）方法,参考[Ribatti et al,1997]5.体外细胞迁移检测:平面单层细胞损伤愈合实验,结合倒置相差显微镜观察,参考[Bürk,1973;Vasvari et al,2007]6.线粒体膜电位（MMP）检测:利用荧光探针（TMRM）并结合激光扫描共聚焦显微术检测,参考[Falchi et al,2005]7.ROS检测:利用荧光探针（DCHF）并结合激光扫描共聚焦显微术检测8.NADPH氧化酶活性检测:参考Li et al的方法[Li et al,2002]9.SOD活性检测:利用SOD检测试剂盒10.NO含量检测:利用NO检测试剂盒11.eNOS活性检测:利用eNOS检测试剂盒12.PC-PLC活性检测:参考吴兴中等人的方法[Wu et al,1997]13.细胞内蛋白分布及表达水平检测:1)免疫细胞化学法结合激光扫描共聚焦显微术,检测P53和NF-κB蛋白的表达水平及分布2)Western blot方法,检测P53、膜整连蛋白β₄和H-ras蛋白的表达水平研究结果1.ABO抑制去除血清和生长因子诱导的血管内皮细胞凋亡在去除血清和FGF-2的条件下,ABO（50-200μM）处理血管内皮细胞24 h,HUVEC的存活率明显升高（图2,p＜0.05）,其最佳浓度为50μM,因此在以下研究中均用该浓度处理血管内皮细胞。用ABO处理细胞12和24 h,凋亡小体明显减少（图3）;吖啶橙染色和TUNEL染色结果表明,ABO明显抑制血管内皮细胞凋亡（图4和图5,p＜0.05）。2.ABO促进血管生成2.1体外Matrigel毛细血管样结构形成实验结果表明,在去除血清和FGF-2的条件下,ABO以时间依赖的方式（24-96 h）明显促进血管生成（图6,p＜0.01）;存在FGF-2（70ng/mL）的条件下,ABO和FGF-2可协同促进血管生成（图7）;仅有存在血清（20%,v/v）的条件下,ABO不能促进血管生成（图8）;血清（20%,v/v）和FGF-2（70ng/mL）都存在的条件下,在培养后期,ABO促进血管生成（图9）。2.2体内鸡胚尿囊膜血管形成实验显示,ABO（20nmol/100μL）促进血管网形成（图10,p＜0.05）。2.3单层细胞损伤愈合实验结果显示,ABO以时间依赖的方式（6-24 h）显著促进细胞迁移（图11,p＜0.01）。3.ABO抑制血管内皮细胞凋亡和促进血管生成的分子机制3.1用ABO处理细胞12和24 h,可以明显降低线粒体膜电位（图12和图13,p＜0.05）和细胞内ROS水平（图14和图15,p＜0.05）。3.2用ABO处理细胞12 h,可以明显降低NADPH氧化酶（图16,p＜0.05）和PC-PLC的活性（图19,p＜0.05）,但对SOD的活性没有影响（图17,p＞0.05）。3.3用ABO处理细胞12 h,明显增强eNOS的活性（图18B,p＜0.05）并增加NO含量（图18A,p＜0.05）,但是处理6和24 h,eNOS的活性和NO释放量没有变化（图18）。3.4免疫细胞化学检测结果表明,用ABO处理细胞6和12 h,NF-κB在细胞质中均匀分布,未观察到NF-κB的核移位现象（图23）;用ABO处理细胞24 h,明显抑制P53蛋白的表达并阻止了P53的核移位（图20,p＜0.05）。3.5 Western Blot检测结果表明,用ABO处理细胞24 h,明显抑制P53蛋白（图21B,p＜0.05）和膜整连蛋白β₄的表达（图22B,p＜0.05）,但是用ABO处理12 h,P53和膜整连蛋白β₄的表达均没有变化（图21A和图22A,p＞0.05）。用ABO处理细胞12和24 h,均明显抑制H-ras蛋白的表达（图24,p＜0.05）。结论1.ABO明显抑制去除血清和生长因子诱导的血管内皮细胞凋亡。2.ABO有效促进血管生成。3.在去除血清和生长因子的条件下,ABO通过稳定线粒体膜电位,防止超极化,维持线粒体的功能,同时通过降低NADPH氧化酶的活性,抑制了细胞内过高的ROS水平而有效抑制了细胞凋亡,并促进了细胞迁移和血管生成。此外,ABO还通过增强eNOS的活性使NO的含量上升,进一步加强血管生成作用。4.ROS、PC-PLC和p53之间有密切联系且均与线粒体相关。ABO很可能通过影响这一条信号转导途径中的相关因子,即控制ROS的水平,抑制PC-PLC的活性以及P53、膜整连蛋白β₄和H-ras蛋白的表达,抑制了去除血清和生长因子诱导的HUVEC凋亡并促进了血管形成。说明ABO是研究血管内皮细胞凋亡、血管形成和两者关联机制的有效工具。更多还原

【Abstract】 BACKGROUND AND OBJECTIVEVascular endothelial cells（VECs）play important roles in vascular development. Numerous studies show that dysregulation of VEC apoptosis leads to vascular dysfunction and is involved in various inflammatory and degenerative diseases.These severe diseases impair our health seriously.At present,how to prevent and cure these diseases is a problem to solve.Angiogenesis refers to the formation of new capillaries from preexisting vessels. Angiogenesis plays essential roles in physiological processes such as embryonic development and in pathologic conditions（e.g.,tumor growth and atherosclerosis）. The process of angiogenesis consists of several signal transductions,which include proliferation,survival,migration,extracellular matrix degradation,differentiation and morphogenesis.Angiogenesis has close relationship with endothelial cell apoptosis. Inhibition of VEC apoptosis providing VEC survival is thought to be an essential issue during vessel formation.The molecular mechanisms associating VEC apoptosis with angiogenesis are not elucidated clearly now.The use of small cell-permeable molecules to effect biological phenomena,also known as chemical genetics,has made a significant impact in diverse areas of biological medicine.The utilization of chemical genetics may discover key factors involved in apoptosis and angiogenesis.Therefore,it can provide experimental evidences to illustrate issues mentioned above.Our previous study confirmed that human umbilical vein endothelial cell（HUVEC）apoptosis was induced by deprived of serum and FGF-2.We synthesized a series of 2,3-dihydro-3-substituted-1,4-benzoxazine derivatives and found that 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine（ABO）could obviously elevate VEC viability in the absence of serum and FGF-2.Consequently we presume that ABO might be an inhibitor of VEC apoptosis as well as a promoter of angiogenesis.In this study,we aim to elucidate the role of ABO in VEC apoptosis and angiogenesis as well as the molecular mechanisms associating endothelial cell apoptosis with angiogenesis utilizing small molecule compound,and provide new insights into the clinical prevention and therapy for blood vessel degenerative diseases.It is well known that redox plays important roles in cell apoptosis and angiogenesis. The components involved in redox include mitochondria,reactive oxygen species （ROS）,NADPH oxidase,superoxide dismutase（SOD）,nitric oxide（NO）and endothelial nitric oxide synthase（eNOS）etc.Accordingly,we first investigate the effect of ABO on signal factors involved in redox.Then,we study the role of ABO in other components closely related to apoptosis and angiogenesis（e.g., Phosphatidylcholine-specific phospholipase C（PC-PLC）,P53,integrinβ₄,nuclear factorκB（NF-κB）and H-ras）.CONTENTS1.Study of ABO on inhibiting vascular endothelial cell apoptosis induced by deprived of serum and FGF-22.Study of ABO on promoting angiogenesis3.Study of molecular mechanisms on ABO inhibiting VEC apoptosis and promoting angiogenesisMETHODS1.Vascular endothelial cell culture:HUVECs were obtained as described previously [Jaffe et al.,1973].2.Cell apoptosis measurement: 2.1 The cell viability was determined by MTT-assay2.2 Observation of cell morphological changes by phase contrast microscope2.3 Analysis of nuclear fragmentation and chromatin condensation by acridine orange staining combined with laser scan confocol microscope2.4 TUNEL assay to determine the apoptotic rate3.Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously[Kureishi Y et al,2000]4.Angiogenesis assay in vivo:chick embryo chorioallantoic membrane（CAM） method as described previously[Ribatti et al,1997]5.Cell migration assay in vitro:monolayer cell wound healing assay as described previously[Bürk,1973;Vasvari et al,2007]6.Mitochondrial membrane potential（MMP）assay:fluorescent probe（TMRM） combined with laser scan confocol microscope as described previously[Falchi et al,2005]7.ROS assay:fluorescent probe（DCHF）combined with laser scan confocol microscope8.NADPH oxidase activity assay as described[Li et al,2002]9.SOD activity assay:SOD detection kit10.NO production assay:NO detection kit11.eNOS activity assay:eNOS detection kit12.PC-PLC activity assay as described[Wu et al.,1997]13.Analysis of cellular distribution and expression of proteins:1)Examination of the changes in P53 and NF-κB protein levels and distributions by immunocytochemistry combined with laser scan confocol microscope2)Analysis the levels of P53、integrinβ₄ and H-ras by Western blot assayRESULTS1.ABO inhibits vascular endothelial cell apoptosis induced by deprived of serum and FGF-2 At 24 h,ABO（50-200μM）significantly increased VEC viability（Fig.2,p＜0.05）. The optimized concentration is 50μM and the following studies use 50μM ABO to treat VECs.At 12 and 24 h,under phase microscope,the number of apoptotic bodies decreased obviously with treatment of ABO（Fig.3）;AO staining showed that ABO inhibited nuclear fragmentation as well as chromatin condensation（Fig.4）.TUNEL assay showed that the apoptotic rate decreased obviously when VECs were exposed to ABOfor 24 h（Fig.5,p＜0.05）.2.ABO promotes angiogenesis2.1 Capillary-like tube formation assay on Matrigel in vitro showed that in the absent of serum and FGF-2,ABO significantly promote angiogenesis in a time-dependent manner（24-96 h）（Fig.6,p＜0.01）;in the presence of FGF-2（70ng/mL）,ABO and FGF-2 could synergistically facilitate angiogenesis（Fig.7）;in the presence of serum （20%,v/v）,ABO could not promote angiogenesis（Fig.8）;in the presence of serum （20%,v/v）and FGF-2（70ng/mL）,ABO facilitated angiogenesis at the late cultural stage（Fig.9）.2.2 CAM assay in vivo showed that ABO（20nmol/100μL）promoted the formation of vascular net（Fig.10,p＜0.05）.2.3 Monolayer cell wound healing assay in vitro showed that ABO（50μM）obviously promoted cell migration in a time-dependent manner（Fig.11,p＜0.01）.3.Molecular mechanism of ABO inhibiting VEC apoptosis and promoting angiogenesis3.1 At 12 and 24 h,ABO significantly decreased mitochondrial membrane potential （Fig.12 & Fig.13,p＜0.05）and cellular ROS level（Fig.14 & Fig.15,p＜0.05）.3.2 At 12 h,ABO significantly decreased the activities of NADPH oxidase（Fig.16, p＜0.05）and PC-PLC（Fig.19,p＜0.05）.ABO has no effect on SOD activity（Fig.17, P＞0.05）.3.3 At 12 h,ABO（50μM）significantly increased the activity of eNOS（Fig.18B, p＜0.05）and NO production（Fig.18A,p＜0.05）.At 6 and 24 h,there is no difference of eNOS activity and NO production between control group and ABO treatment group（Fig.18）. 3.4 Immunocytochemistry assay showed that NF-κB nuclear translocation was not observed when VECs were exposed to ABO for 6 and 12 h（Fig.23）;at 24 h,ABO obviously depressed P53 expression and blocked P53 nuclear translocation（Fig.20, p＜0.05）.3.5 Western Blot assay showed that at 24 h,ABO significantly inhibited P53（Fig.21B, p＜0.05）and integrinβ₄ expression（Fig.22B,p＜0.05）,but ABO have no effect on P53 andβ₄ expression at 12 h（Fig.21A & Fig.22A,p＞0.05）.At 12 and 24 h,ABO obviously depressed H-ras expression（Fig.24,p＜0.05）.CONCLUSION1.ABO significantly inhibited vascular endothelial cell apoptosis induced by deprived of serum and FGF-2.2.ABO effectively promoted angiogenesis.3.In the absence of serum and FGF-2,ABO could maintain mitochondrial functions through stabilizing MMP and preventing hyperpolarization.Moreover,ABO decreased activity of NADPH oxidase.Accordingly,ABO could effectively inhibit VEC apoptosis and promote migration as well as angiogenesis due to depressing high level of cellular ROS.In addition,ABO further enhanced angiogenesis via increasing eNOS activity and NO production.4.ROS had close relationship with PC-PLC and P53,and all of them were associated with mitochondria.ABO probably effected relative factors involved in this signal transduction pathway,that is,controlling ROS levels,depressing PC-PLC activity as well as the expression of P53,integrinβ₄ and H-ras,to inhibit VEC apoptosis induced by deprived of serum and FGF-2 and promote angiogenesis.Our data suggested that ABO was a very useful agent for insights into various questions of angiogenesis and might have a therapeutic potential in vascular diseases.更多还原