【作者】 苑晓晨；

【导师】 修瑞娟；

【作者基本信息】 北京协和医学院 ， 生物医学工程， 2011， 博士

【摘要】 目的：第二部分：为了探讨褪黑素(MEL)对血管单层内皮细胞屏障的保护作用,以培养在Transwell系统内的单层人脐静脉内皮细胞(HUVECs)为模型,观察MEL对IL-1β诱导的单层内皮细胞通透性的影响。第二部分：观察MEL对IL-1β诱导的HUVECs间黏附连接和细胞骨架的影响。第三部分：探讨MEL对单层内皮细胞屏障保护作用可能的受体途径和信号通路。方法：第一部分：原代分离培养HUVECs,并使用包被小鼠抗人CD31单克隆抗体的免疫磁珠对原代分离的HUVECs进一步纯化。以小鼠抗人vWF单克隆抗体及FITC标记的山羊抗小鼠荧光二抗鉴定纯化后的HUVECs。将纯化后的HUVECs接种在Transwell系统,通过检测不同干预条件下透过单层内皮的荧光标记的葡聚糖来反映内皮通透性的改变。第二部分：用免疫荧光显微镜和激光共聚焦显微镜观察不同干预条件下内皮细胞黏附连接蛋白VE钙粘素(VE-cadherin)和细胞骨架蛋白的变化情况。使用Western blot检测VE-cadherin的表达变化情况。第三部分：检测MEL和IL-1p对影响内皮细胞间连接和细胞骨架的重要的信号通路Rho GTPase家族中Rac蛋白的影响；应用MEL受体阻滞剂Luz、Rac的抑制剂NSC-23766,探讨MEL发挥HUVECs屏障保护作用的受体途径和信号通路。结果：第二部分：(1)成功分离培养原代HUVECs,免疫磁珠筛选后能继续贴壁生长并传代,倒置显微镜下HUVECs呈鹅卵石状,vWF免疫荧光染色证实细胞确为HUVECs。细胞呈梭形、圆形或多角形。(2)与空白对照组相比,5、10和20ng/mL的IL-1β分别使HUVECs通透性由1.5±0.1升高为4.8±0.3、5.2±0.4和9.8±0.8(P<0.01)；10和100μmol/L的MEL分别使10ng/mL的IL-1β诱导的通透性从5.2±0.4降至2.8±0.3和2.7±0.3(P<0.01)。此结果与“时间-过程”通透性实验结果一致。第三部分：(1)荧光显微镜观察：正常融合后的内皮细胞表达VE-cadherin呈现出连续不间断的状态；IL-1β(10ng/ml)破坏了细胞间的黏附连接,使VE-cadherin在细胞膜上的表达下调,并出现断裂。MEL(10μmol/L)预处理后,VE-cadherin的表达状态得到了改善。(2)激光共聚焦显微镜下观察：IL-1β(10ng/ml)明显的破坏了细胞间的黏附连接,使正常状态下分布在细胞边缘的肌动蛋白转变为跨细胞体的应力纤维,原本融合的细胞间出现明显的缝隙。MEL(10μmol/L)预处理后,没有出现明显的黏附连破坏现象,细胞中心应力纤维的表达减少,边缘部位的肌动蛋白增加,细胞间缝隙变小。(3)Western Blot结果表明：与对照组相比,IL-1p使HUVECs的VE-cadherin表达量下调。MEL可抑制IL-18诱导的VE-cadherin下调,但仍低于对照组。第三部分：(1)Luz阻断了MEL对内皮屏障的保护作用；阻断了MEL对内皮黏附连接和细胞骨架的影响,导致细胞连接破坏和应力纤维增加,细胞间出现缝隙。(2)MEL预处理抑制了IL-1β诱导的Rac的失活,而Luz可以阻断MEL的这种作用。(3)NSC-23766抑制了MEL对内皮屏障的保护作用。结论：(1)IL-1β诱导HUVECs黏附连接蛋白VE-cadherin表达降低,应力纤维增加,细胞间出现缝隙,通透性升高。(2)MEL通过激活Rac信号途径改善了内皮细胞黏附连接和细胞骨架的状态,抑制了IL-1β诱导的内皮通透性的升高。更多还原

【Abstract】 ObjecivePart one:To determine the effect of melatonin (MEL) on interleukin-1β(IL-1β)-induced impairment of permeability from human umbilical vein endothelial cells (HUVECs). Part two:To determine the effect of MEL on IL-1β-induced damage of endothelial adherens junctions and actin remodeling.Part three:To explore the possible receptors and cell signalling pathways by which melatonin exerts endothelial barrier protective effects.MethodsPart one:Primary HUVECs were isolated from fresh human umbilical veins and cultured after immunomagnetic separation by anti-CD31 antibody coated Dynabeads. For identification of endothelial cells, the cells were incubated with mouse anti-von Willebrand Facto (vWF) and FITC-conjugated goat anti-mouse secondary antibody. The signals were detected by fluorescence microscope. Paracellular permeability was studied in a Transwell system by measuring the flux of FITC-dextran across the endothelial monolayer.Part two:To detect actin and VE-cadherin,the cells were incubated with FITC-conjugated phalloidin and anti-VE-cadherin antibody, then observed by fluorescence microscope and confocal imaging system.Part three:Using Rac activation assays to test the involvement of small GTPase Rac in the melatonin-induced endothelial barrier protective effects as well as cell contact reorganization. Luzindole, MEL membrane receptor antagonist and NSC-23766, a specific inhibitor to Rac were used to explore the possible receptors and cell signalling pathways. ResultsPart one:(1) Primary HUVECs were isolated sucessfully from fresh human umbilical veins and cultured after purified by immunomagnetic separation. The endothelial origin was confirmed by the positive labeling of vWF with immunofluorescence staining. (2) Treatment with IL-1β(5,10 and 20ng/ml) increased the relative permeability of HUVECs from 1.5±0.1 to 4.8±0.3,5.2±0.4 and 9.8±0.8 (P<0.01); MEL (10 and 100μmol/L) attenuated IL-1β(10ng/ml) induced increase of permeability from 5.2±0.4 to 2.8±0.3 and 2.7±0.3 (P<0.01). This result is consistent with the time course analyses of cell permeability.Part two:(1) Fluorescence microscope:By VE-cadherin staining, treatement with IL-1βcaused a clear disruption of adherens junctions. Obvious signs of disruption of adherens junctions after the addition of melatonin in advance were not found. (2) Confocal microscope:A clear change of the actin cytoskeleton is observed from filamentous actin at the cell periphery in normal cells to stress fibers spanning the cell body in the IL-1β-induced cells. Pretreatment with melatonin induced significant reduction in central stress fibers compared with IL-1βtreated cells. (3) Western blotting: The protein level of VE-cadherin was reduced in IL-1β-treated cells, while that of the reduction was attenuated by melatonin.Part three:(1) Luzindole effectively inhibited the protective effect of melatonin on IL-1β-induced increase of endothelial permeability. (2) The inactivation responses of the Rac in HUVECs were investigated by stimulation with IL-1β. Melatonin pretreatment inhibited the IL-1β-induced decrease in the GTPbound form of Rac. Luzindole prevented the inhibitory effects of melatonin on IL-1β-induced Rac inactivation. (3) NSC-23766 abolished the effects of melatonin on IL-1β-induced adherens junctions, cytoskeletal remodeling and the expression of VE-cadherin.Conclusions:Our results provide evidence that L-1βtreatment disrupted HUVECs adherens junctions and induced stress fiber and paracellular gap formation. These changes increased the permeability of the endothelial monolayer. Melatonin inhibits IL-1β-induced endothelial permeability by tightening intercellular junctions and reducing IL-1β-induced stress fiber and paracellular gap formation via activation of Rac.更多还原