白藜蘆醇調(diào)控內(nèi)皮細胞凋亡及促血栓分子P-Selectin、vWF表達的實驗研究
發(fā)布時間：2019-06-28 20:29
【摘要】：DVT是骨科大手術(shù)后常見的臨床并發(fā)癥,屬于靜脈回流障礙性疾病,發(fā)病率高,危害性大,是骨科手術(shù)患者圍手術(shù)期死亡的重要原因之一。DVT發(fā)病機制復雜,涉及內(nèi)皮細胞、血小板、白細胞等多方面因素,近年來研究表明,血管內(nèi)皮細胞損傷,是靜脈血栓形成最重要的原因之一,內(nèi)皮細胞損傷后可釋放各種粘附分子、血小板活化因子、組織因子,增強凝血,促進DVT形成;然而,氧化應激反應、炎癥反應是引起靜脈內(nèi)皮細胞損傷的常見原因,氧化應激、炎癥可引起機體活性氧、炎癥因子產(chǎn)生增加,使細胞脂質(zhì)過氧化、NO減少,從而引起內(nèi)皮細胞損傷、凋亡,促進DVT形成;同時,有研究表明,NF-κB可以通過上調(diào)組織因子表達,促進凝血,從而影響DVT形成;許多研究表明,白藜蘆醇具有抗氧化、抗炎、保護內(nèi)皮細胞損傷的功能,但是,在DVT形成方面卻罕見報道。因此,本研究旨在探討:白藜蘆醇對內(nèi)皮細胞氧化損傷、凋亡的抑制作用,以及對促血栓分子P-Selectin、vWF表達的影響及調(diào)控機制。[目的]1.探討白藜蘆醇對內(nèi)皮細胞氧化損傷、凋亡的影響。2.探討白藜蘆醇對內(nèi)皮細胞損傷后促血栓分子P-Selectin、vWF表達的影響及調(diào)控機制。[方法]本研究分為兩部分:1.第一部分,本實驗部分分為三組:(1)空白對照組;(2)H202組:以20μmo/L的H202處理人臍靜脈內(nèi)皮細胞24小時建立靜脈內(nèi)皮細胞損傷模型;(3)RES+H2O2組:以30μmo/L的白藜蘆醇預處理HUVECs 2小時后,再以200μmo/L的H202處理細胞24小時。采用MTT法檢測各組細胞活力,以探討白藜蘆醇對H202誘導的HUVECs損傷的保護作用;采用熒光探針DCFH-DA捕獲法、激光共聚焦顯微鏡檢測細胞內(nèi)活性氧ROS含量,以探討白藜蘆醇對內(nèi)皮細胞氧化應激的影響;Hoechst 33258染色法、熒光顯微鏡,AnnexinV-FITC/PI雙染法、流式細胞儀檢測細胞凋亡,以探討白藜蘆醇對內(nèi)皮細胞凋亡的影響。2.第二部分,本實驗部分分為四組;(1)空白對照組;(2)H202組:本組與第一部分相同;(3)RES+H2O2組:本組與第一部分相同;(4)BAY11-7082+H2O2組:以5μmol/LNF-κB特異性抑制劑BAY 11-7082預處理細胞4小時后,再加入 200μmol/L H2O2 孵育細胞 24 小時。采用 Real-Time PCR、western blot檢測各組NF-κB、P-Selectin、vWF基因及蛋白表達變化,以探討白藜蘆醇對內(nèi)皮細胞損傷后促血栓分子P-Selectin、vWF表達的影響及調(diào)控機制。[結(jié)果]1.第一部分實驗結(jié)果:采用200μmol/L的H2O2處理HUVECs 24小時,即H202組,與對照組相比,細胞活力明顯降低(P0.01),細胞內(nèi)ROS含量明顯增多(P0.01),細胞凋亡率明顯增加(P0.01);然而采用30μmo/L白藜蘆醇預處理HUVECs 2小時后,再用200μmo/L H202孵育細胞24小時,即RES+H2O2組,與H202組相比,細胞活力明顯增高(P0.01),細胞內(nèi)ROS含量明顯減少(P0.01),細胞凋亡率明顯減少(P0.01)。2.第二部分實驗結(jié)果:(1)用200μmol/L H202處理HUVECs 24小時后,即H202組,其NF-κB、P-Selectin、vWFmRNA及蛋白表達較對照組明顯增高(P0.01)。(2)然而在加入了 NF-κB的特異性抑制劑BAY 11-7082 5μmol/L預處理4小時后,再用200μmol/LH2O2處理細胞24小時,即BAY11-7082+H2O2組,與H2O2組比較,其NF-κBmRNA及蛋白表達出現(xiàn)了下調(diào)(P0.01),P-Selectin、vWFmRNA及蛋白表達也隨之下調(diào)(P0.05)。(3)而在加入30μmol/L的白藜蘆醇預處理2小時后,再用200μmol/LH2O2處理細胞24小時,即RES+H2O2組,與H2O2組比較,其NF-κBmRNA及蛋白表達出現(xiàn)了下調(diào)(P0.01),P-Selectin、vWFmRNA及蛋白表達也隨之下調(diào)(P0.05)。(4)RES+H2O2組與 BAY 11-7082+H2O2組比較,BAY 11-7082+H2O2組NF-κB mRNA及蛋白表達下調(diào)更明顯(P0.01),vWFmRNA及蛋白表達下調(diào)也更明顯(P0.01)。[結(jié)論]1.白藜蘆醇可減少內(nèi)皮細胞損傷、凋亡。2.白藜蘆醇可減少內(nèi)皮細胞損傷后ROS生成。3.NF-κB參與了內(nèi)皮細胞損傷后促血栓分子P-Selectin、vWF表達。4.白藜蘆醇可以抑制內(nèi)皮細胞損傷后促血栓分子P-Selectin、vWF的激活,且可能通過NF-κB信號通路發(fā)揮效應;推測其對DVT形成具有一定的防治作用。
[Abstract]:DVT is a common clinical complication after large-size orthopedic surgery, and it is one of the important causes of the perioperative death in the patients with venous reflux disease, high incidence and high risk. The pathogenesis of the DVT is complicated, and it is related to the factors such as endothelial cell, platelet, and white blood cell. In recent years, the research has shown that the damage of the vascular endothelial cells is one of the most important reasons for venous thrombosis, and the endothelial cells can release various adhesion molecules and platelet activating factors after the endothelial cells are damaged. the tissue factor, the enhanced blood coagulation and the promotion of the formation of the DVT; however, the oxidative stress reaction, the inflammatory reaction is a common cause of the damage of the vein endothelial cells, the oxidative stress and the inflammation can cause the active oxygen and the inflammation factor of the organism to increase, and the lipid peroxidation and the NO of the cells are reduced, so as to cause injury and apoptosis of the endothelial cells and promote the formation of the DVT; at the same time, the research shows that the NF-B can increase the expression of the tissue factor and promote the coagulation, thereby affecting the formation of the DVT, The function of protecting endothelial cell damage, however, is rare in the formation of DVT. Therefore, the purpose of this study is to study the effect of the white and aloe on the oxidative damage and apoptosis of the endothelial cells, as well as the effect of the expression of P-Selectin and vWF on the thrombogenic molecules. [Objective] 1. Objective To study the effect of the white-and-white aloe on the oxidative damage and apoptosis of the endothelial cells. To study the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells. [Method] This study is divided into two parts:1. The first part, this experiment part was divided into three groups: (1) blank control group; (2) H202 group: human umbilical vein endothelial cells were treated with 20. mu. mo/ L H202 for 24 hours to establish a model of vein endothelial cell damage; (3) RES + H2O2 group: after the HUVECs were pre-treated with 30. m The cells were treated with 200. m u.M/ L of H202 for 24 hours. The activity of the cells in each group was detected by MTT method, and the protective effect of the white and aloe on the H202-induced HUVECs injury was discussed. The content of reactive oxygen ROS in the cells was detected by using the fluorescence probe DCFH-DA capture method and the laser confocal microscope. Hoechst 33258 staining, fluorescence microscope, Annexin V-FITC/ PI double staining method and flow cytometry were used to detect the apoptosis of the cells. The second part, this experiment part is divided into four groups; (1) blank control group; (2) H202 group: this group is the same as the first part; (3) RES + H2O2 group: this group is the same as the first part; (4) BAY11-7082 + H2O2 group: after 4 hours of pretreatment of BAY 11-7082 with 5. m The cells were incubated with 200. m u.mol/ L of H2O2 for 24 hours. Using Real-Time PCR and western blot, the expression of NF-B, P-Selectin, vWF and protein in each group were detected, and the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells were discussed. [Results] 1. The results of the first part showed that the cell viability of HUVECs was significantly lower than that of the control group (P0.01). The content of ROS in the cells increased significantly (P0.01), and the rate of apoptosis was significantly increased (P0.01). However, after the pretreatment of HUVECs by 30 & mu; mo/ L, the cells were incubated with 200 & mu; mo/ L H202 for 24 hours, that is, the RES + H2O2 group, and the activity of the cells was significantly higher than that of the H202 group (P0.01). The content of ROS in the cells was significantly decreased (P0.01), and the apoptosis rate of the cells was significantly decreased (P0.01). The results of the second part: (1) After the treatment of HUVECs for 24 hours with 200 & mu; mol/ L H202, the expression of NF-B, P-Selectin, vWFmRNA and protein in the H202 group was significantly higher than that in the control group (P0.01). (2) After the pretreatment of BAY 11-7082 5umol/ L, the specific inhibitor BAY 11-7082 5 & mu; mol/ L was treated with 200 & mu; mol/ L H _ 2O _ 2 for 24 hours, that is, BAY11-7082 + H2O2 group, and the expression of NF-BmRNA and protein of BAY11-7082 + H2O2 group was down-regulated (P0.01), P-Selectin, The expression of vWFmRNA and protein was also down-regulated (P0.05). (3) After the pre-treatment of 30. m u.mol/ L of Phragon for 2 hours, the cells were treated with 200. m u.mol/ L H _ 2O _ 2 for 24 hours, that is, the RES + H2O2 group, and the expression of NF-BmRNA and protein in the group was down-regulated (P0.01), and the expression of P-Selectin, vWFmRNA and protein was also down-regulated (P0.05). (4) Compared with BAY 11-7082 + H2O2 group, the expression of NF-EMAB mRNA and protein in BAY 11-7082 + H2O2 group was lower than that of BAY 11-7082 + H2O2 group (P0.01). [Conclusion] 1. Phragon can reduce the damage and apoptosis of the endothelial cells. 3. NF-EMAB was involved in the expression of P-Selectin and vWF after endothelial cell injury. It can inhibit the activation of P-Selectin and vWF, and may play an important role in the formation of DVT.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R68
本文編號：2507590
[Abstract]:DVT is a common clinical complication after large-size orthopedic surgery, and it is one of the important causes of the perioperative death in the patients with venous reflux disease, high incidence and high risk. The pathogenesis of the DVT is complicated, and it is related to the factors such as endothelial cell, platelet, and white blood cell. In recent years, the research has shown that the damage of the vascular endothelial cells is one of the most important reasons for venous thrombosis, and the endothelial cells can release various adhesion molecules and platelet activating factors after the endothelial cells are damaged. the tissue factor, the enhanced blood coagulation and the promotion of the formation of the DVT; however, the oxidative stress reaction, the inflammatory reaction is a common cause of the damage of the vein endothelial cells, the oxidative stress and the inflammation can cause the active oxygen and the inflammation factor of the organism to increase, and the lipid peroxidation and the NO of the cells are reduced, so as to cause injury and apoptosis of the endothelial cells and promote the formation of the DVT; at the same time, the research shows that the NF-B can increase the expression of the tissue factor and promote the coagulation, thereby affecting the formation of the DVT, The function of protecting endothelial cell damage, however, is rare in the formation of DVT. Therefore, the purpose of this study is to study the effect of the white and aloe on the oxidative damage and apoptosis of the endothelial cells, as well as the effect of the expression of P-Selectin and vWF on the thrombogenic molecules. [Objective] 1. Objective To study the effect of the white-and-white aloe on the oxidative damage and apoptosis of the endothelial cells. To study the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells. [Method] This study is divided into two parts:1. The first part, this experiment part was divided into three groups: (1) blank control group; (2) H202 group: human umbilical vein endothelial cells were treated with 20. mu. mo/ L H202 for 24 hours to establish a model of vein endothelial cell damage; (3) RES + H2O2 group: after the HUVECs were pre-treated with 30. m The cells were treated with 200. m u.M/ L of H202 for 24 hours. The activity of the cells in each group was detected by MTT method, and the protective effect of the white and aloe on the H202-induced HUVECs injury was discussed. The content of reactive oxygen ROS in the cells was detected by using the fluorescence probe DCFH-DA capture method and the laser confocal microscope. Hoechst 33258 staining, fluorescence microscope, Annexin V-FITC/ PI double staining method and flow cytometry were used to detect the apoptosis of the cells. The second part, this experiment part is divided into four groups; (1) blank control group; (2) H202 group: this group is the same as the first part; (3) RES + H2O2 group: this group is the same as the first part; (4) BAY11-7082 + H2O2 group: after 4 hours of pretreatment of BAY 11-7082 with 5. m The cells were incubated with 200. m u.mol/ L of H2O2 for 24 hours. Using Real-Time PCR and western blot, the expression of NF-B, P-Selectin, vWF and protein in each group were detected, and the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells were discussed. [Results] 1. The results of the first part showed that the cell viability of HUVECs was significantly lower than that of the control group (P0.01). The content of ROS in the cells increased significantly (P0.01), and the rate of apoptosis was significantly increased (P0.01). However, after the pretreatment of HUVECs by 30 & mu; mo/ L, the cells were incubated with 200 & mu; mo/ L H202 for 24 hours, that is, the RES + H2O2 group, and the activity of the cells was significantly higher than that of the H202 group (P0.01). The content of ROS in the cells was significantly decreased (P0.01), and the apoptosis rate of the cells was significantly decreased (P0.01). The results of the second part: (1) After the treatment of HUVECs for 24 hours with 200 & mu; mol/ L H202, the expression of NF-B, P-Selectin, vWFmRNA and protein in the H202 group was significantly higher than that in the control group (P0.01). (2) After the pretreatment of BAY 11-7082 5umol/ L, the specific inhibitor BAY 11-7082 5 & mu; mol/ L was treated with 200 & mu; mol/ L H _ 2O _ 2 for 24 hours, that is, BAY11-7082 + H2O2 group, and the expression of NF-BmRNA and protein of BAY11-7082 + H2O2 group was down-regulated (P0.01), P-Selectin, The expression of vWFmRNA and protein was also down-regulated (P0.05). (3) After the pre-treatment of 30. m u.mol/ L of Phragon for 2 hours, the cells were treated with 200. m u.mol/ L H _ 2O _ 2 for 24 hours, that is, the RES + H2O2 group, and the expression of NF-BmRNA and protein in the group was down-regulated (P0.01), and the expression of P-Selectin, vWFmRNA and protein was also down-regulated (P0.05). (4) Compared with BAY 11-7082 + H2O2 group, the expression of NF-EMAB mRNA and protein in BAY 11-7082 + H2O2 group was lower than that of BAY 11-7082 + H2O2 group (P0.01). [Conclusion] 1. Phragon can reduce the damage and apoptosis of the endothelial cells. 3. NF-EMAB was involved in the expression of P-Selectin and vWF after endothelial cell injury. It can inhibit the activation of P-Selectin and vWF, and may play an important role in the formation of DVT.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R68
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