【摘要】：研究背景:動脈粥樣硬化(As)是一個復(fù)雜的病理生理過程,目前認(rèn)為其發(fā)病主要與血漿中脂蛋白水平升高,動脈內(nèi)膜損傷導(dǎo)致動脈壁通透性增加以及脂蛋白穿過單層內(nèi)皮細(xì)胞在內(nèi)膜下沉積有關(guān)。臨床研究發(fā)現(xiàn),氧化脂蛋白(a)(oxLp(a))在As斑塊中廣泛存在,其中破裂斑塊尤其突出,并且與泡沫細(xì)胞共存,這提示了oxLp(a)與As的發(fā)生發(fā)展密切相關(guān)。血管內(nèi)皮在維持血管的正常形態(tài)和功能中起重要作用,血管內(nèi)皮是由單層內(nèi)皮細(xì)胞緊密連接而成,因此細(xì)胞與細(xì)胞之間的連接至關(guān)重要,而橋粒連接是最主要的血管內(nèi)皮的連接方式。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,參與了血管壁生理屏障的構(gòu)成,維持細(xì)胞與細(xì)胞之間的完整性,因此細(xì)胞與細(xì)胞之間的這種橋粒連接在調(diào)控單層內(nèi)皮細(xì)胞的通透性上起著非常重要的作用。本研究旨在探討oxLp(a)對人臍靜脈內(nèi)皮細(xì)胞DSG1和DSC2表達(dá)的影響,以及由此導(dǎo)致的內(nèi)皮細(xì)胞通透性的改變,以及活性氧(ROS)在其中的作用。 實驗一:oxLp(a)對人臍靜脈內(nèi)皮細(xì)胞DSG1,DSC2表達(dá)的影響 目的:觀察oxLp(a)對人臍靜脈內(nèi)皮細(xì)胞上跨膜蛋白DSG1和DSC2表達(dá)的影響。方法:待細(xì)胞生長融合后,每次實驗前6h給HUVECs換上新鮮的無血清培養(yǎng)基以獲得同步化生長,6h后再換上新鮮的培養(yǎng)基并加入不同濃度的oxLp(a)(0mg/L,25 mg/L,50 mg/L,100 mg/L)處理HUVECs24h,檢測不同濃度的oxLp(a)對HUVECs上DSG1,DSC2 mRNA以及蛋白表達(dá)的影響;再用100 mg/L oxLp(a)分別處理HUVECs不同時間(0h,6h,12h,24h),用RT-PCR和Western blotting分別檢測DSG1,DSC2 mRNA以及蛋白的表達(dá)。 結(jié)果:oxLp(a)濃度依賴性地下調(diào)HUVECs上DSG1,DSC2 mRNA以及蛋白的表達(dá),oxLp(a)在25 mg/L時即有顯著作用(P0.05),且隨著oxLp(a)濃度的升高,DSG1、DSC2 mRNA和蛋白表達(dá)水平呈濃度依賴的下降趨勢,100 mg/L組與0mg/L相比,DSG1、DSC2 mRNA和蛋白的表達(dá)下調(diào)最明顯(P0.05);100 mg/L oxLp(a)在孵育6h即能顯著下調(diào)DSG1,DSC2 mRNA以及蛋白的表達(dá)(P0.05),且隨著處理時間的延長,DSG1、DSC2 mRNA和蛋白表達(dá)水平呈時間依賴的下降趨勢,24h組與0h組比較,DSG1、DSC2 mRNA和蛋白的表達(dá)下調(diào)最明顯(P0.05)。 實驗二:ROS參與oxLp(a)對HUVECs DSG1,DSC2表達(dá)的調(diào)節(jié) 目的:觀察oxLp(a)是否通過促進(jìn)細(xì)胞內(nèi)活性氧的生成來調(diào)節(jié)HUVECs上DSG1,DSC2的表達(dá)。 方法:用100 mg/L Lp(a)、100 mg/L oxLp(a)、100 mg/L BSA、200μmol/L H_2O_2分別與HUVECs孵育24h,空白組作為對照,用RT-PCR和Western blotting分別檢測DSG1,DSC2 mRNA以及蛋白的表達(dá)情況。用100 mg/L oxLp(a)、100 mg/LSOD預(yù)孵育1h后再加入100 mg/L oxLp(a)、100 mg/LSOD分別處理HUVECs24h,空白組作為對照,用DCFH-DA熒光探針檢測細(xì)胞內(nèi)ROS的生成情況;用RT-PCR和Western blotting分別檢測DSG1,DSC2 mRNA以及蛋白的表達(dá)。 結(jié)果:oxLp(a)、H_2O_2顯著下調(diào)HUVECs DSG1,DSC2 mRNA以及蛋白的表達(dá)(P0.05),而Lp(a)、BSA對其無明顯影響(P0.05)。oxLp(a)組與空白對照組相比,細(xì)胞內(nèi)活性氧的生成明顯增多,用SOD預(yù)孵育后,活性氧的產(chǎn)生有所降低,而SOD組與對照組相比無明顯差別;oxLp(a)抑制DSG1,DSC2 mRNA以及蛋白的表達(dá)(P0.05),而用SOD預(yù)孵育后,這種抑制作用得到了改善,SOD組與對照組相比無明顯差別,這與活性氧的測定結(jié)果是相一致的。 實驗三:oxLp(a)對單層內(nèi)皮細(xì)胞通透性的影響 目的:觀察oxLp(a)對單層內(nèi)皮細(xì)胞通透性的影響。 方法:用100 mg/L oxLp(a)處理HUVECs24h后,在上室加入FITC-dextran,分別在不同的時間點(0h,0.25h,0.5h,1h,2h,4h)收集下室中的液體,測量液體的熒光強(qiáng)度,確定測量通透性的最佳時間。用不同濃度的oxLp(a()0mg/L,25 mg/L,50 mg/L,100 mg/L)分別或者與SOD共同孵育HUVECs 24h后,在上室中加入FITC- dextran與孵育2h,空白組作為對照,取下室中的液體用熒光分光光度計測定熒光強(qiáng)度,代表通透性改變。 結(jié)果:oxLp(a)處理細(xì)胞24h后,在上室中加入FITC-dextran,不同時間點收集下室中的液體測量熒光強(qiáng)度,結(jié)果顯示,在2h時熒光強(qiáng)度最強(qiáng),4h與2h相比無顯著差異(P0.05),說明2h是測量通透性的最佳時間;oxLp(a)也以劑量依賴性的方式增加單層內(nèi)皮細(xì)胞的通透性,在25 mg/L時即有作用(P0.05),隨著oxLp(a)濃度的升高,單層內(nèi)皮細(xì)胞的通透性也逐漸增加,100 mg/L時作用最明顯(P0.05);100 mg/L oxLp(a)所致的單層內(nèi)皮細(xì)胞通透性的增加能夠被SOD所抑制(P0.05)。結(jié)論:oxLp(a)以劑量和時間依賴性方式下調(diào)HUVECs上DSG1,DSC2的表達(dá),并增加單層內(nèi)皮細(xì)胞的通透性;活性氧生成增加與oxLp(a)致DSG1,DSC2表達(dá)的下調(diào)以及單層內(nèi)皮細(xì)胞通透性的上升有關(guān)。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張志輝;李旭平;周勝華;祁述善;;CD40配體、妊娠相關(guān)血漿蛋白A和丙二醛化低密度脂蛋白的變化與冠狀動脈斑塊破裂相關(guān)性[J];中國動脈硬化雜志;2007年02期

相關(guān)碩士學(xué)位論文 前1條

1 李媛彬;oxLDL干擾HUVECs的DSG1和DSC2表達(dá)并增加單層內(nèi)皮細(xì)胞對LDL的通透性[D];南華大學(xué);2010年

，

本文編號：2234655