oxLDL干扰HUVECs的DSG1和DSC2表达并增加单层内皮细胞对LDL的通透性
发布时间：2019-03-13 15:58
【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(P0.05)。 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(P0.05);SOD能使50mg/L oxLDL诱导的HUVECs单层通透性降低(P0.05)。 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(P0.05),而LDL、BSA对其无明显影响(P0.05)。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(P0.05),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。
[Abstract]:Background of the study: With regard to the occurrence of atherosclerosis (AS), it is generally believed that the increase in the level of lipoprotein in plasma, the increase in the permeability of the arterial wall due to the injury of the vascular endothelial cells, and the subcutaneous deposition of the lipoproteins through the endothelial barrier, etc. Oxidized low-density lipoprotein (oxLDL) was detected in atherosclerotic (AS) plaque and foam cells, suggesting that oxLDL was closely related to the development of AS. But to date, no reports of the effect of oxLDL on the mechanism of LDL permeability have been found. The bridge core glycoprotein-1 (DSG1) and the bridge-particle core-gel protein-2 (DSC2) belong to the member of the bridge-particle calcium-binding protein family, and the bridge particle core glycoprotein-1 (DSG1) and the bridge-particle core-binding protein-2 (DSC2) exist in the vascular endothelial cell gap, and the physical barrier function between the blood vessel wall and the blood is exerted to maintain the integrity of the cell and the cell. Therefore, the connection between the endothelial cells and the cells plays an important role in the regulation of permeability. The purpose of this study is to study the effect of xLDL on the expression of the endothelial cells DSG1 and DSC2, as well as the effect on the permeability of the endothelial cells. experimental one oxLDL on HUVEC cells DSG1 and DSC2 Objective: To observe the effect of oxLDL on the expression of cross-membrane protein DSG1 and DSC2 on human umbilical vein endothelial cells, and to the monolayer of human umbilical vein endothelial cells after the stimulation of this oxidized lipoprotein. Change of cell permeability:24 hours prior to each experiment, the HUVECs were changed fresh HUVEC was treated with different concentrations of oxLDL (0 mg/ L,10 mg/ L,25 mg/ L,50 mg/ L), and the expression of DSG1, DSC2 mRNA and protein was detected. The different time of HUVEC was treated with 50 mg/ L oxLDL (0 h,6 h,12 h,24 h), and the cells DSG1 and DSC were respectively detected by Western blotting and RT-PCR. The results showed that the expression of DG1, DSC2 mRNA and protein in HUVECs was down regulated by the expression of DG1, DSC2 mRNA and protein. Inter-and dose-dependent relationship (P0.05). Experiment 2 The effect of oxLDL on the monolayer permeability of HUVECs: view The effect of oxLDL on the single-layer permeability of human umbilical vein endothelial cells was investigated by using oxLDL at different concentrations (0 mg/ L,10 mg/ L,25 mg/ L,50 mg/ L) or with SOD, respectively, HUVECs24h and blank group were used as control, and the permeability of single-layer cells to BSA and LDL was detected by Transwell system. The content of FITC-BSA and FITC-LDL was determined by spectrophotometry. The single-layer permeability of HUVECs induced by g/ L oxLDL decreased (P0.05). Objective: To study the effect of three ROS in the regulation of the expression of human HUVEC DSG1 and DSC2: the study of oxL The expression of DG1 and DSC2 in HUVEC cells was regulated by using LDL (50 mg/ L), oxLDL (50 mg/ L), BSA (100 mg/ L) and H2O2 (5 mg/ L) respectively. The expression level of HUVECs DSG1 and DSC2 mRNA and protein was detected by ern blotting, and the expression levels of the mRNA and protein of HUVECs DSG1 and DSC2 were detected with 50 mg/ L of oxLDL,50 mg/ L SOD, and oxLDL and 50 mg/ L SOD were respectively incubated with HUVECs for 24 h, and the production of active oxygen in the cells was detected by DCFH-DA staining. The expression level of DSC2 mRNA and protein was observed by laser confocal microscope. The results showed that the expression of oxLDL and H2O2 in HUVECs DSG1, DSC2 mRNA and protein was down-regulated (P0.05), and LDL and BSA had no significant effect on the expression of active oxygen in the cells (P0.05). DSG1,DSC2 mRNA Conclusion: OxLDL has the effects of interfering with the expression of DG1 and DSC2 and increasing the expression of DSC1 and DSC2.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2439540
[Abstract]:Background of the study: With regard to the occurrence of atherosclerosis (AS), it is generally believed that the increase in the level of lipoprotein in plasma, the increase in the permeability of the arterial wall due to the injury of the vascular endothelial cells, and the subcutaneous deposition of the lipoproteins through the endothelial barrier, etc. Oxidized low-density lipoprotein (oxLDL) was detected in atherosclerotic (AS) plaque and foam cells, suggesting that oxLDL was closely related to the development of AS. But to date, no reports of the effect of oxLDL on the mechanism of LDL permeability have been found. The bridge core glycoprotein-1 (DSG1) and the bridge-particle core-gel protein-2 (DSC2) belong to the member of the bridge-particle calcium-binding protein family, and the bridge particle core glycoprotein-1 (DSG1) and the bridge-particle core-binding protein-2 (DSC2) exist in the vascular endothelial cell gap, and the physical barrier function between the blood vessel wall and the blood is exerted to maintain the integrity of the cell and the cell. Therefore, the connection between the endothelial cells and the cells plays an important role in the regulation of permeability. The purpose of this study is to study the effect of xLDL on the expression of the endothelial cells DSG1 and DSC2, as well as the effect on the permeability of the endothelial cells. experimental one oxLDL on HUVEC cells DSG1 and DSC2 Objective: To observe the effect of oxLDL on the expression of cross-membrane protein DSG1 and DSC2 on human umbilical vein endothelial cells, and to the monolayer of human umbilical vein endothelial cells after the stimulation of this oxidized lipoprotein. Change of cell permeability:24 hours prior to each experiment, the HUVECs were changed fresh HUVEC was treated with different concentrations of oxLDL (0 mg/ L,10 mg/ L,25 mg/ L,50 mg/ L), and the expression of DSG1, DSC2 mRNA and protein was detected. The different time of HUVEC was treated with 50 mg/ L oxLDL (0 h,6 h,12 h,24 h), and the cells DSG1 and DSC were respectively detected by Western blotting and RT-PCR. The results showed that the expression of DG1, DSC2 mRNA and protein in HUVECs was down regulated by the expression of DG1, DSC2 mRNA and protein. Inter-and dose-dependent relationship (P0.05). Experiment 2 The effect of oxLDL on the monolayer permeability of HUVECs: view The effect of oxLDL on the single-layer permeability of human umbilical vein endothelial cells was investigated by using oxLDL at different concentrations (0 mg/ L,10 mg/ L,25 mg/ L,50 mg/ L) or with SOD, respectively, HUVECs24h and blank group were used as control, and the permeability of single-layer cells to BSA and LDL was detected by Transwell system. The content of FITC-BSA and FITC-LDL was determined by spectrophotometry. The single-layer permeability of HUVECs induced by g/ L oxLDL decreased (P0.05). Objective: To study the effect of three ROS in the regulation of the expression of human HUVEC DSG1 and DSC2: the study of oxL The expression of DG1 and DSC2 in HUVEC cells was regulated by using LDL (50 mg/ L), oxLDL (50 mg/ L), BSA (100 mg/ L) and H2O2 (5 mg/ L) respectively. The expression level of HUVECs DSG1 and DSC2 mRNA and protein was detected by ern blotting, and the expression levels of the mRNA and protein of HUVECs DSG1 and DSC2 were detected with 50 mg/ L of oxLDL,50 mg/ L SOD, and oxLDL and 50 mg/ L SOD were respectively incubated with HUVECs for 24 h, and the production of active oxygen in the cells was detected by DCFH-DA staining. The expression level of DSC2 mRNA and protein was observed by laser confocal microscope. The results showed that the expression of oxLDL and H2O2 in HUVECs DSG1, DSC2 mRNA and protein was down-regulated (P0.05), and LDL and BSA had no significant effect on the expression of active oxygen in the cells (P0.05). DSG1,DSC2 mRNA Conclusion: OxLDL has the effects of interfering with the expression of DG1 and DSC2 and increasing the expression of DSC1 and DSC2.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
【引证文献】
中国硕士学位论文全文数据库 前1条
1 张晓蕾;氧化脂蛋白（a）对HUVECs的DSG1和DSC2表达及单层内皮细胞通透性的影响[D];南华大学;2011年
，本文编号：2439540
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