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
发布时间：2018-09-10 14:05
【摘要】：研究背景:动脉粥样硬化(As)是一个复杂的病理生理过程,目前认为其发病主要与血浆中脂蛋白水平升高,动脉内膜损伤导致动脉壁通透性增加以及脂蛋白穿过单层内皮细胞在内膜下沉积有关。临床研究发现,氧化脂蛋白(a)(oxLp(a))在As斑块中广泛存在,其中破裂斑块尤其突出,并且与泡沫细胞共存,这提示了oxLp(a)与As的发生发展密切相关。血管内皮在维持血管的正常形态和功能中起重要作用,血管内皮是由单层内皮细胞紧密连接而成,因此细胞与细胞之间的连接至关重要,而桥粒连接是最主要的血管内皮的连接方式。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,参与了血管壁生理屏障的构成,维持细胞与细胞之间的完整性,因此细胞与细胞之间的这种桥粒连接在调控单层内皮细胞的通透性上起着非常重要的作用。本研究旨在探讨oxLp(a)对人脐静脉内皮细胞DSG1和DSC2表达的影响,以及由此导致的内皮细胞通透性的改变,以及活性氧(ROS)在其中的作用。 实验一:oxLp(a)对人脐静脉内皮细胞DSG1,DSC2表达的影响 目的:观察oxLp(a)对人脐静脉内皮细胞上跨膜蛋白DSG1和DSC2表达的影响。方法:待细胞生长融合后,每次实验前6h给HUVECs换上新鲜的无血清培养基以获得同步化生长,6h后再换上新鲜的培养基并加入不同浓度的oxLp(a)(0mg/L,25 mg/L,50 mg/L,100 mg/L)处理HUVECs24h,检测不同浓度的oxLp(a)对HUVECs上DSG1,DSC2 mRNA以及蛋白表达的影响;再用100 mg/L oxLp(a)分别处理HUVECs不同时间(0h,6h,12h,24h),用RT-PCR和Western blotting分别检测DSG1,DSC2 mRNA以及蛋白的表达。 结果:oxLp(a)浓度依赖性地下调HUVECs上DSG1,DSC2 mRNA以及蛋白的表达,oxLp(a)在25 mg/L时即有显著作用(P0.05),且随着oxLp(a)浓度的升高,DSG1、DSC2 mRNA和蛋白表达水平呈浓度依赖的下降趋势,100 mg/L组与0mg/L相比,DSG1、DSC2 mRNA和蛋白的表达下调最明显(P0.05);100 mg/L oxLp(a)在孵育6h即能显著下调DSG1,DSC2 mRNA以及蛋白的表达(P0.05),且随着处理时间的延长,DSG1、DSC2 mRNA和蛋白表达水平呈时间依赖的下降趋势,24h组与0h组比较,DSG1、DSC2 mRNA和蛋白的表达下调最明显(P0.05)。 实验二:ROS参与oxLp(a)对HUVECs DSG1,DSC2表达的调节 目的:观察oxLp(a)是否通过促进细胞内活性氧的生成来调节HUVECs上DSG1,DSC2的表达。 方法:用100 mg/L Lp(a)、100 mg/L oxLp(a)、100 mg/L BSA、200μmol/L H_2O_2分别与HUVECs孵育24h,空白组作为对照,用RT-PCR和Western blotting分别检测DSG1,DSC2 mRNA以及蛋白的表达情况。用100 mg/L oxLp(a)、100 mg/LSOD预孵育1h后再加入100 mg/L oxLp(a)、100 mg/LSOD分别处理HUVECs24h,空白组作为对照,用DCFH-DA荧光探针检测细胞内ROS的生成情况;用RT-PCR和Western blotting分别检测DSG1,DSC2 mRNA以及蛋白的表达。 结果:oxLp(a)、H_2O_2显著下调HUVECs DSG1,DSC2 mRNA以及蛋白的表达(P0.05),而Lp(a)、BSA对其无明显影响(P0.05)。oxLp(a)组与空白对照组相比,细胞内活性氧的生成明显增多,用SOD预孵育后,活性氧的产生有所降低,而SOD组与对照组相比无明显差别;oxLp(a)抑制DSG1,DSC2 mRNA以及蛋白的表达(P0.05),而用SOD预孵育后,这种抑制作用得到了改善,SOD组与对照组相比无明显差别,这与活性氧的测定结果是相一致的。 实验三:oxLp(a)对单层内皮细胞通透性的影响 目的:观察oxLp(a)对单层内皮细胞通透性的影响。 方法:用100 mg/L oxLp(a)处理HUVECs24h后,在上室加入FITC-dextran,分别在不同的时间点(0h,0.25h,0.5h,1h,2h,4h)收集下室中的液体,测量液体的荧光强度,确定测量通透性的最佳时间。用不同浓度的oxLp(a()0mg/L,25 mg/L,50 mg/L,100 mg/L)分别或者与SOD共同孵育HUVECs 24h后,在上室中加入FITC- dextran与孵育2h,空白组作为对照,取下室中的液体用荧光分光光度计测定荧光强度,代表通透性改变。 结果:oxLp(a)处理细胞24h后,在上室中加入FITC-dextran,不同时间点收集下室中的液体测量荧光强度,结果显示,在2h时荧光强度最强,4h与2h相比无显著差异(P0.05),说明2h是测量通透性的最佳时间;oxLp(a)也以剂量依赖性的方式增加单层内皮细胞的通透性,在25 mg/L时即有作用(P0.05),随着oxLp(a)浓度的升高,单层内皮细胞的通透性也逐渐增加,100 mg/L时作用最明显(P0.05);100 mg/L oxLp(a)所致的单层内皮细胞通透性的增加能够被SOD所抑制(P0.05)。结论:oxLp(a)以剂量和时间依赖性方式下调HUVECs上DSG1,DSC2的表达,并增加单层内皮细胞的通透性;活性氧生成增加与oxLp(a)致DSG1,DSC2表达的下调以及单层内皮细胞通透性的上升有关。
[Abstract]:BACKGROUND: Atherosclerosis (As) is a complex pathophysiological process. It is presently believed that atherosclerosis is mainly caused by elevated plasma lipoprotein levels, increased arterial wall permeability due to arterial intimal injury, and deposition of lipoprotein through monolayer endothelial cells. OxLp (a) is closely related to the development of As. Vascular endothelium plays an important role in maintaining the normal morphology and function of blood vessels. Vascular endothelium is formed by the tight junction of monolayer endothelial cells, so the connection between cells is related. Desmosome-linked glycoprotein-1 (DSG1) and desmosome-linked protein-2 (DSC2) are members of the desmosome cadherin family, which are involved in the formation of the vascular wall physiological barrier and maintain the integrity between cells. Therefore, desmosome-linked glycoprotein-1 (DSG1) and desmosome-2 (DSC2) are regulated between cells. The purpose of this study was to investigate the effects of oxLp (a) on the expression of DSG1 and DSC2 in human umbilical vein endothelial cells, the changes of endothelial cell permeability and the role of reactive oxygen species (ROS).
Experiment 1: the effect of oxLp (a) on the expression of DSG1 and DSC2 in human umbilical vein endothelial cells
OBJECTIVE: To observe the effect of oxLp (a) on the expression of transmembrane proteins DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured in fresh serum-free medium 6 hours before each experiment to obtain synchronized growth. After 6 hours, fresh medium was added and oxLp (a) (0 mg/L, 25 mg/L, 50 mg/L, 100 mg/L) was added. The effects of oxLp(a) on the expression of DSG1, DSC2 mRNA and protein in HUVECs were detected after 24 hours of treatment, and the expression of DSG1, DSC2 mRNA and protein in HUVECs were detected by RT-PCR and Western blotting after 100 mg/L oxLp(a) treatment at different times (0 h, 6 h, 12 h, 24 h).
Results: OxLp (a) down-regulated the expression of DSG1, DSC2 mRNA and protein in HUVECs in a concentration-dependent manner. OxLp (a) had a significant effect at 25 mg/L (P 0.05). With the increase of oxLp (a) concentration, the expression of DSG1, DSC2 mRNA and protein decreased in a concentration-dependent manner. Significant (P 0.05); 100 mg/L oxLp (a) significantly decreased the expression of DSG1, DSC2 mRNA and protein at 6 h of incubation (P 0.05), and the expression of DSG1, DSC2 mRNA and protein decreased in a time-dependent manner with the prolongation of incubation time. Compared with 0 h group, the expression of DSG1, DSC2 mRNA and protein decreased most significantly at 24 h (P 0.05).
Experiment two: ROS was involved in the regulation of HUVECs DSG1 and DSC2 expression by oxLp (a).
AIM: To investigate whether oxLp (a) regulates the expression of DSG1 and DSC2 on HUVECs by promoting the production of reactive oxygen species (ROS).
METHODS: The expression of DSG1, DSC2 mRNA and protein was detected by RT-PCR and Western blotting, respectively. After incubation with 100 mg/L oxLp (a), 100 mg/L oxLp (a), 100 mg/L BSA, 200 umol/L H_2O_2 and HUVECs for 24 hours, the blank group was treated with 100 mg/L oxLp (a), 100 mg/L OD for 1 hour and 100 mg/L oxLp (a), respectively. Cs24h, blank group as control, DCFH-DA fluorescent probe was used to detect the production of ROS, and RT-PCR and Western blotting were used to detect the expression of DSG1, DSC2 mRNA and protein.
Results: OxLp (a) and H_2O_2 significantly decreased the expression of HUVECs DSG1, DSC2 mRNA and protein (P 0.05), while Lp (a) and BSA had no significant effect on it (P 0.05). Compared with the blank control group, the production of reactive oxygen species (ROS) in the cells of OxLp (a) group increased significantly, and the production of ROS decreased after SOD incubation, but there was no significant difference between the SOD group and the control group. The expression of DSG1, DSC2 mRNA and protein was inhibited (P 0.05). After incubation with SOD, the inhibition was improved. There was no significant difference between SOD group and control group, which was consistent with the results of reactive oxygen species determination.
Experiment three: effect of oxLp (a) on permeability of monolayer endothelial cells
Objective: To observe the effect of oxLp (a) on the permeability of monolayer endothelial cells.
METHODS: After treating HUVECs with 100 mg/L oxLp (a) for 24 hours, FITC-dextran was added to the room to collect the liquid in the lower chamber at different time points (0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h) respectively. The fluorescence intensity of the liquid was measured to determine the best time for measuring the permeability. After incubation of HUVECs for 24 hours, FITC-dextran was added into the upper chamber and incubated for 2 hours. The blank group was used as the control group. Fluorescence intensity of the liquid in the lower chamber was measured by fluorescence spectrophotometer, representing the change of permeability.
Results: After 24 hours of oxLp (a) treatment, FITC-dextran was added into the upper chamber to measure the fluorescence intensity. The results showed that the fluorescence intensity was the strongest at 2 hours, and there was no significant difference between 4 hours and 2 hours (P 0.05), indicating that 2 hours was the best time to measure the permeability; oxLp (a) also increased the monolayer endothelial fineness in a dose-dependent manner. The permeability of monolayer endothelial cells increased with the increase of oxLp (a) concentration at 25 mg/L (P 0.05). The effect was most obvious at 100 mg/L (P 0.05). The increase of permeability of monolayer endothelial cells induced by 100 mg/L oxLp (a) could be inhibited by SOD (P 0.05). Conclusion: OxLp (a) can be inhibited in a dose-and time-dependent manner. The expression of DSG1 and DSC2 on HUVECs was down-regulated and the permeability of monolayer endothelial cells was increased. The increase of reactive oxygen species production was related to the down-regulation of DSG1 and DSC2 expression induced by oxLp(a) and the increase of permeability of monolayer endothelial cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2234655
[Abstract]:BACKGROUND: Atherosclerosis (As) is a complex pathophysiological process. It is presently believed that atherosclerosis is mainly caused by elevated plasma lipoprotein levels, increased arterial wall permeability due to arterial intimal injury, and deposition of lipoprotein through monolayer endothelial cells. OxLp (a) is closely related to the development of As. Vascular endothelium plays an important role in maintaining the normal morphology and function of blood vessels. Vascular endothelium is formed by the tight junction of monolayer endothelial cells, so the connection between cells is related. Desmosome-linked glycoprotein-1 (DSG1) and desmosome-linked protein-2 (DSC2) are members of the desmosome cadherin family, which are involved in the formation of the vascular wall physiological barrier and maintain the integrity between cells. Therefore, desmosome-linked glycoprotein-1 (DSG1) and desmosome-2 (DSC2) are regulated between cells. The purpose of this study was to investigate the effects of oxLp (a) on the expression of DSG1 and DSC2 in human umbilical vein endothelial cells, the changes of endothelial cell permeability and the role of reactive oxygen species (ROS).
Experiment 1: the effect of oxLp (a) on the expression of DSG1 and DSC2 in human umbilical vein endothelial cells
OBJECTIVE: To observe the effect of oxLp (a) on the expression of transmembrane proteins DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured in fresh serum-free medium 6 hours before each experiment to obtain synchronized growth. After 6 hours, fresh medium was added and oxLp (a) (0 mg/L, 25 mg/L, 50 mg/L, 100 mg/L) was added. The effects of oxLp(a) on the expression of DSG1, DSC2 mRNA and protein in HUVECs were detected after 24 hours of treatment, and the expression of DSG1, DSC2 mRNA and protein in HUVECs were detected by RT-PCR and Western blotting after 100 mg/L oxLp(a) treatment at different times (0 h, 6 h, 12 h, 24 h).
Results: OxLp (a) down-regulated the expression of DSG1, DSC2 mRNA and protein in HUVECs in a concentration-dependent manner. OxLp (a) had a significant effect at 25 mg/L (P 0.05). With the increase of oxLp (a) concentration, the expression of DSG1, DSC2 mRNA and protein decreased in a concentration-dependent manner. Significant (P 0.05); 100 mg/L oxLp (a) significantly decreased the expression of DSG1, DSC2 mRNA and protein at 6 h of incubation (P 0.05), and the expression of DSG1, DSC2 mRNA and protein decreased in a time-dependent manner with the prolongation of incubation time. Compared with 0 h group, the expression of DSG1, DSC2 mRNA and protein decreased most significantly at 24 h (P 0.05).
Experiment two: ROS was involved in the regulation of HUVECs DSG1 and DSC2 expression by oxLp (a).
AIM: To investigate whether oxLp (a) regulates the expression of DSG1 and DSC2 on HUVECs by promoting the production of reactive oxygen species (ROS).
METHODS: The expression of DSG1, DSC2 mRNA and protein was detected by RT-PCR and Western blotting, respectively. After incubation with 100 mg/L oxLp (a), 100 mg/L oxLp (a), 100 mg/L BSA, 200 umol/L H_2O_2 and HUVECs for 24 hours, the blank group was treated with 100 mg/L oxLp (a), 100 mg/L OD for 1 hour and 100 mg/L oxLp (a), respectively. Cs24h, blank group as control, DCFH-DA fluorescent probe was used to detect the production of ROS, and RT-PCR and Western blotting were used to detect the expression of DSG1, DSC2 mRNA and protein.
Results: OxLp (a) and H_2O_2 significantly decreased the expression of HUVECs DSG1, DSC2 mRNA and protein (P 0.05), while Lp (a) and BSA had no significant effect on it (P 0.05). Compared with the blank control group, the production of reactive oxygen species (ROS) in the cells of OxLp (a) group increased significantly, and the production of ROS decreased after SOD incubation, but there was no significant difference between the SOD group and the control group. The expression of DSG1, DSC2 mRNA and protein was inhibited (P 0.05). After incubation with SOD, the inhibition was improved. There was no significant difference between SOD group and control group, which was consistent with the results of reactive oxygen species determination.
Experiment three: effect of oxLp (a) on permeability of monolayer endothelial cells
Objective: To observe the effect of oxLp (a) on the permeability of monolayer endothelial cells.
METHODS: After treating HUVECs with 100 mg/L oxLp (a) for 24 hours, FITC-dextran was added to the room to collect the liquid in the lower chamber at different time points (0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h) respectively. The fluorescence intensity of the liquid was measured to determine the best time for measuring the permeability. After incubation of HUVECs for 24 hours, FITC-dextran was added into the upper chamber and incubated for 2 hours. The blank group was used as the control group. Fluorescence intensity of the liquid in the lower chamber was measured by fluorescence spectrophotometer, representing the change of permeability.
Results: After 24 hours of oxLp (a) treatment, FITC-dextran was added into the upper chamber to measure the fluorescence intensity. The results showed that the fluorescence intensity was the strongest at 2 hours, and there was no significant difference between 4 hours and 2 hours (P 0.05), indicating that 2 hours was the best time to measure the permeability; oxLp (a) also increased the monolayer endothelial fineness in a dose-dependent manner. The permeability of monolayer endothelial cells increased with the increase of oxLp (a) concentration at 25 mg/L (P 0.05). The effect was most obvious at 100 mg/L (P 0.05). The increase of permeability of monolayer endothelial cells induced by 100 mg/L oxLp (a) could be inhibited by SOD (P 0.05). Conclusion: OxLp (a) can be inhibited in a dose-and time-dependent manner. The expression of DSG1 and DSC2 on HUVECs was down-regulated and the permeability of monolayer endothelial cells was increased. The increase of reactive oxygen species production was related to the down-regulation of DSG1 and DSC2 expression induced by oxLp(a) and the increase of permeability of monolayer endothelial cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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1 张志辉;李旭平;周胜华;祁述善;;CD40配体、妊娠相关血浆蛋白A和丙二醛化低密度脂蛋白的变化与冠状动脉斑块破裂相关性[J];中国动脉硬化杂志;2007年02期
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1 李媛彬;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[D];南华大学;2010年
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