利用小分子化合物研究血管内皮细胞凋亡和血管生成的分子机制

发布时间：2018-04-29 16:53

本文选题：血管内皮细胞 + 凋亡　；参考：《山东大学》2008年博士论文

【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VA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β_4、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β_4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β_4的表达(图22B,p＜0.05),但是用ABO处理12 h,P53和膜整连蛋白β_4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β_4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。
[Abstract]:Background and purpose of research
Vascular endothelial cells (VEC) play an important role in the development of blood vessels. A large number of experimental results show that abnormal vascular function caused by vascular endothelial cell apoptosis will cause a variety of inflammatory and degenerative diseases. These major diseases seriously harm human health, and how to prevent and control these diseases is a problem to be solved urgently.
New vascular formation (angiogenesis) plays a key role in physiological processes (such as embryonic development) and pathological processes (such as tumor growth, atherosclerosis). New vascular formation is closely related to endothelial cell apoptosis. Inhibition of vascular endothelial cell apoptosis is a key factor in the promotion of new vascular formation. However, the molecular mechanisms associated with them are currently involved. It is not clear yet.
From the perspective of chemical genetics, small molecular compounds can be used to detect new key factors involved in the apoptosis and angiogenesis of vascular endothelial cells, and thus provide experimental evidence to clarify the scientific problems mentioned above. Previous studies have shown that the removal of serum and growth factors can induce apoptosis of human umbilical vein endothelial cells (HUVEC), and in this condition, 6- ammonia Base -2,3- two hydroxy -3- hydroxymethyl -1,4- benzo VA derivatives (ABO) can increase the survival rate of vascular endothelial cells. Therefore, ABO may be an inhibitor of vascular endothelial cell apoptosis and angiogenesis promoter.
The purpose of this study is to study the apoptosis, angiogenesis and the molecular mechanism of vascular endothelial cells from the perspective of chemical genetics, with small molecular compound ABO as a tool for the prevention and treatment of vascular degenerative diseases.
The known redox reaction (redox) plays an important role in cell apoptosis and angiogenesis, and the components involved in redox include mitochondria, reactive oxygen species (ROS), NADPH oxidase, superoxide dismutase (SOD) and nitric oxide (NO) / endothelial nitric oxide synthase (eNOS). Therefore, this paper first studied the redox signal molecule of ABO. The changes in other molecules that are closely related to apoptosis and angiogenesis, such as phosphatidylcholine specific phospholipase C (PC-PLC), P53, membrane protein beta _4, nuclear factor kappa B (NF- kappa B) and H-ras, are subsequently studied under the action of ABO.
research contents
1.ABO inhibits vascular endothelial cell apoptosis induced by serum and growth factors
The role of 2.ABO in promoting angiogenesis
Molecular mechanism of 3.ABO inhibiting apoptosis and promoting angiogenesis in vascular endothelial cells
research method
1. endothelial cell culture: extraction and culture of human umbilical vein endothelial cells by reference to Jaffe EA et al [Jaffe EA et al 1973]
2. detection of cell apoptosis:
1) MTT detection of cell viability
2) observation of cell morphological changes by inverted phase contrast microscope
3) acridine orange staining combined with laser scanning confocal microscopy to observe nuclear agglutination and fragmentation.
4) TUNEL method, detection of apoptosis rate
3. in vitro angiogenesis detection: Matrigel method, reference [Kureishi Y et al, 2000]
4. detection of blood vessel formation in vivo: chicken embryo allantoic membrane (CAM) method, refer to [Ribatti et al, 1997]
5. in vitro cell migration assay: planar monolayer cell injury healing experiment, combined with inverted phase contrast microscope, with reference to [B u rk, 1973; Vasvari et al, 2007]
6. mitochondrial membrane potential (MMP) detection: fluorescence probe (TMRM) combined with laser scanning confocal microscopy, reference [Falchi et al, 2005]
7.ROS detection: fluorescence probe (DCHF) combined with laser scanning confocal microscopy.
8.NADPH oxidase activity detection: reference Li et al method [Li et al, 2002]
9.SOD activity detection: using SOD detection kit
10.NO content detection: using NO detection kit
11.eNOS activity detection: using eNOS detection kit
12.PC-PLC activity detection: reference to Wu Xingzhong et al. Method [Wu et al, 1997]
The distribution and expression level of protein in 13. cells were detected.
1) immunocytochemistry and laser scanning confocal microscopy were used to detect the expression level and distribution of P53 and NF- kappa B protein.
2) Western blot method was used to detect the expression level of P53, _4 and H-ras protein.
Research results
1.ABO inhibits serum and growth factor induced apoptosis of vascular endothelial cells
Under the conditions of removing serum and FGF-2, ABO (50-200 mu M) treated vascular endothelial cells 24 h, and the survival rate of HUVEC increased significantly (Figure 2, P < 0.05). The optimum concentration was 50 mu M. Therefore, the concentration of vascular endothelial cells was treated with this concentration in the following studies. The apoptotic bodies were significantly reduced by ABO treatment of 12 and 24 h (Fig. 3); acridine orange staining and TUNEL staining. The results showed that ABO significantly inhibited the apoptosis of vascular endothelial cells (FIGS. 4 and 5, P < 0.05).
2.ABO promotes angiogenesis
2.1 in vitro Matrigel capillary like structure formation experimental results showed that, under the condition of removing serum and FGF-2, the time dependent manner (24-96 h) significantly promoted angiogenesis (Figure 6, P < 0.01); under the condition of FGF-2 (70ng/mL), ABO and FGF-2 could promote angiogenesis (Fig. 7); only under the presence of serum (20%, v/v), ABO was not Promoting angiogenesis (Fig. 8); in serum (20%, v/v) and FGF-2 (70ng/mL), ABO promoted angiogenesis at the later stage of culture (Fig. 9).
2.2 angiogenesis experiments in chicken chorioallantoic membrane showed that ABO (20nmol/100 L) promoted the formation of vascular network (FIG. 10, P < 0.05).
2.3 the single cell injury healing experiment showed that ABO significantly promoted cell migration in a time-dependent manner (6-24 h) (FIG. 11, P < 0.01).
Molecular mechanism of 3.ABO inhibiting apoptosis and promoting angiogenesis in vascular endothelial cells
3.1 treatment of cells 12 and 24 h with ABO significantly reduced mitochondrial membrane potential (Figs 12 and 13, P < 0.05) and intracellular ROS level (Figs 14 and 15, P < 0.05).
3.2 cell 12 h treated with ABO could significantly reduce the activity of NADPH oxidase (Figure 16, P < 0.05) and PC-PLC (Fig. 19, P < 0.05), but did not affect the activity of SOD (FIG. 17, P > 0.05).
3.3 the cell 12 h treated with ABO significantly enhanced the activity of eNOS (Figure 18B, P < 0.05) and increased the content of NO (Fig. 18A, P < 0.05), but the activity of 6 and 24 h, eNOS activity and NO release did not change (Fig. 18).
3.4 the results of immunocytochemical detection showed that the cell 6 and 12 h were treated with ABO, NF- kappa B was evenly distributed in the cytoplasm, and the nuclear shift of NF- kappa B was not observed (Fig. 23); the expression of 24 h by ABO was used to inhibit the expression of P53 protein and prevent the nuclear shift of P53 (FIG. 20, P < 0.05).
The results of 3.5 Western Blot test showed that the expression of P53 protein (21B, P < 0.05) and the expression of membrane protein beta _4 were obviously inhibited by ABO treatment of 24 h (Fig. 22B, P < 0.05), but there was no change in the expression of 12 h. Expression (Figure 24, P < 0.05).
conclusion
1.ABO significantly inhibited the apoptosis of vascular endothelial cells induced by serum and growth factor.
2.ABO effectively promotes angiogenesis.
3. under the conditions of removing serum and growth factors, ABO can prevent hyperpolarization and maintain mitochondrial function by stabilizing mitochondrial membrane potential, and reducing the high ROS level by reducing the activity of NADPH oxidase, which effectively inhibits apoptosis and promotes cell migration and angiogenesis. In addition, ABO is also enhanced by eNOS. The activity increased the content of NO and further enhanced angiogenesis.
The close links between 4.ROS, PC-PLC and p53 and mitochondrial related.ABO are likely to affect the level of ROS, the inhibition of the activity of PC-PLC, and the expression of P53, the expression of the membrane protein beta _4 and H-ras protein, which inhibit the removal of HUVEC apoptosis induced by the removal of serum and growth factors and promote the promotion of the apoptosis of HUVEC. Angiogenesis indicates that ABO is an effective tool for studying endothelial cell apoptosis, angiogenesis and their correlation mechanisms.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

【参考文献】