oxLDL干擾HUVECs的DSG1和DSC2表達(dá)并增加單層內(nèi)皮細(xì)胞對(duì)LDL的通透性
發(fā)布時(shí)間：2019-03-13 15:58
【摘要】： 研究背景:關(guān)于動(dòng)脈粥樣硬化(AS)的發(fā)生,目前普遍認(rèn)為與血漿中脂蛋白水平的升高,血管內(nèi)皮細(xì)胞的損傷導(dǎo)致的動(dòng)脈壁通透性增加以及脂蛋白穿過內(nèi)皮屏障在內(nèi)皮下沉積等有關(guān)。在動(dòng)脈粥樣硬化(AS)斑塊和泡沫細(xì)胞中檢測(cè)到了氧化修飾的低密度脂蛋白(oxLDL),提示oxLDL與AS的發(fā)生發(fā)展密切相關(guān)。但迄今為止未見oxLDL促進(jìn)內(nèi)皮細(xì)胞對(duì)LDL通透性機(jī)制方面的報(bào)道。橋粒芯糖蛋白-1(DSG1)和橋粒芯膠蛋白-2(DSC2)屬于橋粒鈣粘素蛋白家族成員,存在于血管內(nèi)皮細(xì)胞間隙,發(fā)揮了血管壁與血液間的物理屏障作用,維持細(xì)胞與細(xì)胞之間的完整性。因此內(nèi)皮細(xì)胞與細(xì)胞間的這種橋粒連接在通透性調(diào)控方面起了重要的作用。本研究意在探討xLDL對(duì)內(nèi)皮細(xì)胞DSG1和DSC2表達(dá)的影響,以及由此導(dǎo)致對(duì)內(nèi)皮細(xì)胞通透性的影響。 實(shí)驗(yàn)一oxLDL對(duì)HUVEC細(xì)胞DSG1,DSC2表達(dá)的影響 目的:觀察oxLDL對(duì)人臍靜脈內(nèi)皮細(xì)胞上跨膜蛋白DSG1和DSC2表達(dá)的影響,以及人臍靜脈內(nèi)皮細(xì)胞受到這種氧化型脂蛋白刺激后對(duì)單層細(xì)胞通透性的變化。 方法:每次實(shí)驗(yàn)前24小時(shí),給HUVECs換新鮮培養(yǎng)基培養(yǎng)后加處理因素。不同濃度oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)處理HUVEC24h,檢測(cè)細(xì)胞DSG1,DSC2 mRNA和蛋白的表達(dá),再用50mg/L oxLDL分別處理HUVEC不同時(shí)間(0h,6h,12h,24h),用Western blotting和RT- PCR分別檢測(cè)細(xì)胞DSG1,DSC2 mRNA和蛋白的表達(dá)。 結(jié)果:HUVECs上有DSG1、DSC2 mRNA與蛋白質(zhì)的表達(dá);oxLDL使DSG1、DSC2 mRNA與蛋白的表達(dá)下調(diào),且成時(shí)間與劑量依賴關(guān)系(P0.05)。 實(shí)驗(yàn)二oxLDL對(duì)HUVECs單層通透性的影響 目的:觀察oxLDL對(duì)人臍靜脈內(nèi)皮細(xì)胞單層通透性的影響。 方法:用不同濃度的oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)分別或者與SOD共同孵育HUVECs24h,空白組作為對(duì)照,通過Transwell系統(tǒng)檢測(cè)單層細(xì)胞對(duì)BSA和LDL的通透性情況,采用熒光分光光度法測(cè)定FITC-BSA和FITC-LDL的含量。 結(jié)果:oxLDL能顯著增加HUVECs單層對(duì)BSA和LDL的通透性,且作用隨著濃度增加而增強(qiáng),在50mg/L時(shí)有顯著作用(P0.05);SOD能使50mg/L oxLDL誘導(dǎo)的HUVECs單層通透性降低(P0.05)。 實(shí)驗(yàn)三ROS參與oxLDL對(duì)HUVEC DSG1和DSC2表達(dá)的調(diào)節(jié) 目的:探討oxLDL是否通過促進(jìn)細(xì)胞內(nèi)ROS生成參與調(diào)節(jié)HUVEC細(xì)胞DSG1和DSC2的表達(dá)。 方法:用LDL(50mg/L)、oxLDL(50mg/L)、BSA(100mg/L)、H2O2(5mg/L)分別與HUVECs孵育24h,空白組作為對(duì)照,用RT-PCR與Western blotting分別檢測(cè)HUVECs DSG1、DSC2 mRNA與蛋白質(zhì)的表達(dá)水平。用50mg/L的oxLDL、50mg/LSOD預(yù)處理再加oxLDL、50mg/LSOD分別與HUVECs孵育24h,用DCFH-DA染色法檢測(cè)細(xì)胞內(nèi)活性氧的生成情況;用RT-PCR與Western blotting分別檢測(cè)HUVECs DSG1、DSC2 mRNA與蛋白質(zhì)的表達(dá)水平;用激光共聚焦顯微鏡觀察細(xì)胞DSG1的免疫反應(yīng)性。 結(jié)果:oxLDL、H2O2使HUVECs DSG1、DSC2 mRNA與蛋白質(zhì)的表達(dá)下調(diào)(P0.05),而LDL、BSA對(duì)其無明顯影響(P0.05)。oxLDL增加細(xì)胞內(nèi)活性氧的生成和降低DSG1、DSC2 mRNA與蛋白質(zhì)的表達(dá)(P0.05),同時(shí)降低HUVECs DSG1免疫反應(yīng)性,且SOD均能抑制上述結(jié)果。 結(jié)論:oxLDL具有干擾DSG1和DSC2的表達(dá)并增加血管內(nèi)皮對(duì)LDL的通透性的作用;活性氧的產(chǎn)生增加可能是oxLDL所介導(dǎo)的單層內(nèi)皮細(xì)胞通透作用的途徑之一。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363
【引證文獻(xiàn)】
中國(guó)碩士學(xué)位論文全文數(shù)據(jù)庫(kù) 前1條
1 張曉蕾;氧化脂蛋白（a）對(duì)HUVECs的DSG1和DSC2表達(dá)及單層內(nèi)皮細(xì)胞通透性的影響[D];南華大學(xué);2011年
，本文編號(hào)：2439540
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