【作者】 李媛彬；

【导师】 易光辉；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 研究背景:关于动脉粥样硬化(AS)的发生,目前普遍认为与血浆中脂蛋白水平的升高,血管内皮细胞的损伤导致的动脉壁通透性增加以及脂蛋白穿过内皮屏障在内皮下沉积等有关。在动脉粥样硬化(AS)斑块和泡沫细胞中检测到了氧化修饰的低密度脂蛋白(oxLDL),提示oxLDL与AS的发生发展密切相关。但迄今为止未见oxLDL促进内皮细胞对LDL通透性机制方面的报道。桥粒芯糖蛋白-1(DSG1)和桥粒芯胶蛋白-2(DSC2)属于桥粒钙粘素蛋白家族成员,存在于血管内皮细胞间隙,发挥了血管壁与血液间的物理屏障作用,维持细胞与细胞之间的完整性。因此内皮细胞与细胞间的这种桥粒连接在通透性调控方面起了重要的作用。本研究意在探讨xLDL对内皮细胞DSG1和DSC2表达的影响,以及由此导致对内皮细胞通透性的影响。实验一oxLDL对HUVEC细胞DSG1,DSC2表达的影响目的:观察oxLDL对人脐静脉内皮细胞上跨膜蛋白DSG1和DSC2表达的影响,以及人脐静脉内皮细胞受到这种氧化型脂蛋白刺激后对单层细胞通透性的变化。方法:每次实验前24小时,给HUVECs换新鲜培养基培养后加处理因素。不同浓度oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)处理HUVEC24h,检测细胞DSG1,DSC2 mRNA和蛋白的表达,再用50mg/L oxLDL分别处理HUVEC不同时间(0h,6h,12h,24h),用Western blotting和RT- PCR分别检测细胞DSG1,DSC2 mRNA和蛋白的表达。结果:HUVECs上有DSG1、DSC2 mRNA与蛋白质的表达;oxLDL使DSG1、DSC2 mRNA与蛋白的表达下调,且成时间与剂量依赖关系(P<0.05)。实验二oxLDL对HUVECs单层通透性的影响目的:观察oxLDL对人脐静脉内皮细胞单层通透性的影响。方法:用不同浓度的oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)分别或者与SOD共同孵育HUVECs24h,空白组作为对照,通过Transwell系统检测单层细胞对BSA和LDL的通透性情况,采用荧光分光光度法测定FITC-BSA和FITC-LDL的含量。结果:oxLDL能显著增加HUVECs单层对BSA和LDL的通透性,且作用随着浓度增加而增强,在50mg/L时有显著作用(P<0.05);SOD能使50mg/L oxLDL诱导的HUVECs单层通透性降低(P<0.05)。实验三ROS参与oxLDL对HUVEC DSG1和DSC2表达的调节目的:探讨oxLDL是否通过促进细胞内ROS生成参与调节HUVEC细胞DSG1和DSC2的表达。方法:用LDL(50mg/L)、oxLDL(50mg/L)、BSA(100mg/L)、H2O2(5mg/L)分别与HUVECs孵育24h,空白组作为对照,用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平。用50mg/L的oxLDL、50mg/LSOD预处理再加oxLDL、50mg/LSOD分别与HUVECs孵育24h,用DCFH-DA染色法检测细胞内活性氧的生成情况;用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平;用激光共聚焦显微镜观察细胞DSG1的免疫反应性。结果:oxLDL、H2O2使HUVECs DSG1、DSC2 mRNA与蛋白质的表达下调(P<0.05),而LDL、BSA对其无明显影响(P>0.05)。oxLDL增加细胞内活性氧的生成和降低DSG1、DSC2 mRNA与蛋白质的表达(P<0.05),同时降低HUVECs DSG1免疫反应性,且SOD均能抑制上述结果。结论:oxLDL具有干扰DSG1和DSC2的表达并增加血管内皮对LDL的通透性的作用;活性氧的产生增加可能是oxLDL所介导的单层内皮细胞通透作用的途径之一。更多还原

【Abstract】 BACKGROUNDThe mechanism of the atherosclerosis (As), is now generally believed closely associated with the increased of lipoproteins level. The increase of endothelial permeability of the arterial vessel wall is favorite for lipoprotein deposit in subendothelial space. Studies has showed that the presence of oxidatively modified low-density lipoprotein (oxLDL) detected in the intima of atherosclerotic plaques and foam cells. It is believed that the oxLDL is closely related to As. But so far,it has not been clearly understood if the oxLDL promotes the endothelium permeability to LDL. Both desmoglein1(DSG1) and desmocollin2 (DSC2) are the member of cadherins. They function at the site of interface of the endothelial cells, and play a role of the vessel walls’physical barriers and maintaining the integrity of the cell to cell. The desmosomal junction of the cell-cell in the permeability regulation may play an important role. this study is to explore the effects of oxLDL on expression of DSG1and DSC2 in endothelial cells, the effect of permeability injury induced by oxidized lipoprotein, and possible mechanisms of these.Part I. Effects of oxLDL on DSG1,DSC2 expression in HUVECsObjective: To identify the effects of oxLDL on DSG1 and DSC2 expression in HUVECs.Methods: Replaced with fresh medium every 24 hours before treatments. The different concentrations of oxLDL (0 mg/L,10 mg/L,25 mg/L,50 mg/L) were incubated with HUVECs for 24h, or with 50mg/L of oxLDL for different times (0h, 6h,12h and 24h). RT-PCR and Western blotting were applied to determine the DSG1 and DSC2 expression in mRNA and protein, respectively.Results: We found that oxLDL decreased DSG1 and DSC2 in both protein and mRNA levels in dose-dependent and time-dependent manners (P<0.05)..Part II. Effects of oxLDL on monolayer permeability in HUVECsObjective: To investigate the effects of oxLDL on monolayer permeability in HUVECs.Methods: HUVECs were cultured in DMEM medium, and replaced with fresh medium every 24 hours before treatments. Microscope was used to observe the monolayer information. HUVECs cultured in upper compartment of transwell were treated by oxLDL(50mg/L) and/or SOD(50mg/L) for 24 hours. And then, The LDL was added in upper compartment for 4 hours until to test the content of the LDL in the lower compartment. This effect represented change of the monolayer permeability to LDL.Results: The oxidized lipoprotein promoted the endothelial monolayer permeability to LDL(P<0.05). Superoxide dismutase(SOD) partly inhibited the effects of oxLDL on monolayer barrier damage(P<0.05).PartⅢ. ROS involved in oxLDL regulation of DSG1 and DSC2 expression in HUVECsObjective: To explore the role of ROS in the regulation of expression on DSG1 and DSC2 induced by oxLDL in HUVECs.Methods: HUVECs were incubated with oxLDL(50mg/L) and LDL(50mg/L), BSA(100mg/L), H2O(25mg/L), respectively, for 24h. control group was designed to PBS incubation. HUVECs were pretreated for 1h with 50mg/L superoxide dismutase(SOD), and then were incubated for another 24h with 50mg/l of oxLDL. Intracellular ROS production was detected by Dichlorofluorescin diacetate (DCFH-DA) dye. The mRNA and protein expression of DSG1and DSC2 was determined by RT- PCR and Western blotting, respectively. Immunoreactivity of DSG1 was detected by laser scanning confocal microscope (LSCM).Results: oxLDL and H2O2 decreased expression of DSG1, DSC2, both mRNA and protein, in HUVECs(P<0.05) , while detected effects were not observed in treatment groups by LDL and BSA. oxLDL increased generation of cellular reactive oxygen species, and reduced expression of DSG1, DSC2 mRNA and protein(P<0.05). Furthermore, oxLDL reduced immunostaining of DSG1 in HUVECs. The results also showed this effect was inhibited by SOD supplement.Conclusions :These findings suggest that oxLDL may interfere the expression of desmosome proteins, DSG1 and DSC2, by which endothelial permeability to LDL is increased. Production of reactive oxygen species(ROS) may be involved in the promotion of monolayer permeability by oxLDL.更多还原