靜脈高壓導致的剪應力改變在AIBP調(diào)控VEGF表達中的作用
發(fā)布時間：2018-11-16 07:37
【摘要】：研究背景:硬腦膜動靜脈瘺(dural arteriovenous fistula,DAVF)是指硬腦膜及其附屬物(大腦鐮、小腦幕)上的動脈與腦皮層靜脈或靜脈竇間發(fā)生的異常血管短路,持續(xù)性的靜脈高壓會引發(fā)腦出血、腦水腫、靜脈性梗死、認知功能下降等神經(jīng)功能障礙。在臨床上,復雜性DAVF仍然難以通過外科治療得到完全治愈,明確該病發(fā)生發(fā)展的原因尤為關鍵。在對DAVF的病因學的研究中,發(fā)現(xiàn)靜脈高壓導致的血管新生起到關鍵作用,因此明確血管新生在硬腦膜動靜脈瘺發(fā)病機制中的作用,成為控制該類疾病進展的重點。實驗目的:1.建立不同靜脈端壓力梯度的大鼠模型,探索靜脈端壓力改變對上矢狀竇剪應力的影響。2.檢測AIBP-VEGF介導的血管新生通路中相關蛋白,在不同靜脈高壓大鼠模型硬腦膜中、在不同剪應力人臍靜脈內(nèi)皮細胞上清液中的表達情況,探索靜脈高壓造成的剪應力改變對AIBP調(diào)控血管新生的影響。實驗方法:1.第一部分:建立不同靜脈壓力梯度的大鼠模型,將80只SD大鼠隨機分為4組,每組20只,其中A組20只大鼠行右側頸總動脈頸外靜脈吻合術+左側橫竇出口閉塞術;B組20只大鼠行右側頸總動脈頸外靜脈吻合術;C組20只大鼠行右側頸外靜脈結扎術及右側頸總動脈結扎術;D組20只大鼠為假手術組,所有大鼠于術后行頂骨磨除術,暴露上矢狀竇,測量并計算3個月后上矢狀竇剪應力,處死大鼠后,取硬膜組織,檢測AIBP、VEGF的表達情況。2.第二部分:按剪應力不同將人臍靜脈內(nèi)皮細胞分為ABCDE四組,根據(jù)第一部分大鼠上矢狀竇剪應力結果,分別于10、5、2.5、1、0dyn/cm2剪應力環(huán)境下培養(yǎng)人臍靜脈內(nèi)皮細胞24小時,取細胞上清行AIBP及VEGF的表達檢測,取細胞檢測細胞總膽固醇的含量,該部分實驗每組各重復進行三次。實驗結果:1.第一部分:A組大鼠上矢狀竇剪應力為5.32±0.31dyn/cm2,B組大鼠上矢狀竇剪應力平均值2.39±0.03dyn/cm2,C組大鼠上矢狀竇剪應力平均值0.97±0.01dyn/cm2,D組大鼠上矢狀竇剪應力平均值0.88±0.01dyn/cm2,A、B組剪應力高于D組,有統(tǒng)計學意義,C、D兩組無統(tǒng)計學意義。Western blot及real-time PCR結果為A、B組大鼠硬腦膜中AIBP的表達明顯降低,VEGF表達明顯增高,與D組之間有統(tǒng)計學差異,C、D組無統(tǒng)計學意義。免疫組化結果可見AIBP在血管全層均有表達。第二部分:A、B、C組高剪應力培養(yǎng)下的臍靜脈內(nèi)皮細胞上清液中,AIBP表達量較E組降低,VEGF表達量較E組升高,P0.05;A、B、C組細胞總膽固醇含量較E組表達量升高,P0.05。D組細胞上清中檢測蛋白及細胞內(nèi)膽固醇總含量和E組相比,均無統(tǒng)計學意義。實驗結論:1.SD大鼠靜脈高壓模型建立成功3個月后,上矢狀竇的剪應力增高,硬膜血管內(nèi)皮中AIBP表達下降。2.高剪應力條件下培養(yǎng)的人臍靜脈內(nèi)皮細胞AIBP的表達下調(diào),細胞內(nèi)總膽固醇增加,VEGF表達上調(diào)。提示剪應力可能通過調(diào)節(jié)膽固醇代謝,調(diào)控VEGF/VEGFR通道,影響血管新生。
[Abstract]:Background: dural arteriovenous fistula (dural arteriovenous fistula,DAVF) is an abnormal short circuit between the artery on the dura mater and its appendages (falx cerebelli, tentorium cerebellum) and the cortical vein or venous sinus. Persistent venous hypertension can lead to cerebral hemorrhage, cerebral edema, venous infarction, cognitive impairment and other neurological disorders. In clinic, complex DAVF is still difficult to be completely cured by surgical treatment. In the study of the etiology of DAVF, it is found that angiogenesis caused by venous hypertension plays a key role. Therefore, it is important to clarify the role of angiogenesis in the pathogenesis of dural arteriovenous fistula and to control the progress of this disease. Objective: 1. A rat model with different venous pressure gradient was established to explore the effect of venous pressure change on the shear stress of superior sagittal sinus. 2. The expression of related proteins in AIBP-VEGF mediated angiogenesis pathway and in the supernatant of human umbilical vein endothelial cells under different shear stress was detected in the dura mater of rats with different venous hypertension. To explore the effect of shear stress changes caused by venous hypertension on angiogenesis regulated by AIBP. Experimental method: 1. Part one: the rat models with different venous pressure gradient were established. 80 SD rats were randomly divided into 4 groups (20 rats in each group). Group A (20 rats) underwent right common carotid jugular vein anastomosis (right common carotid jugular vein anastomosis) and left transverse sinus outlet occlusion. The right common carotid artery external jugular vein anastomosis was performed in 20 rats in group B, the right external jugular vein ligation and the right common carotid artery ligation were performed in 20 rats in group C. In group D, 20 rats were sham-operated. All rats underwent parietal bone removal after operation, exposed the superior sagittal sinus, measured and calculated the shear stress of the superior sagittal sinus 3 months later, killed the rats, took the dural tissue and detected the expression of AIBP,VEGF. 2. The second part: human umbilical vein endothelial cells were divided into four groups according to shear stress. According to the results of shear stress of the superior sagittal sinus of rats in the first part, human umbilical vein endothelial cells were cultured under the shear stress of 10 ~ (5) ~ 2 ~ (2.5) ~ (10) dyn / cm ~ (2) for 24 hours, respectively. The expression of AIBP and VEGF were detected in the supernatant of the cells and the total cholesterol content in the cells was measured. The experiment was repeated three times in each group. Experimental results: 1. Part one: the average shear stress of superior sagittal sinus in group A was 5.32 鹵0.31 dyn / cm ~ (-2) in group B, and the mean value was 2.39 鹵0.03 dyn / cm ~ (2) in group C, and 0.97 鹵0.01 dyn / cm ~ (-2) in group C, respectively. The average shear stress of superior sagittal sinus in group D was 0.88 鹵0.01dyn / cm ~ 2 路m ~ (-2). The shear stress in group B was higher than that in group D (P < 0.05), but there was no significant difference between group C and D (. Western blot and real-time PCR). The expression of AIBP and VEGF in the dura mater of group B was significantly lower than that in group D, but there was no significant difference between group D and group C. Immunohistochemical results showed that AIBP was expressed in the whole vascular layer. The second part: in the supernatant of umbilical vein endothelial cells cultured under high shear stress, the expression of AIBP was lower than that of group E, and the expression of VEGF was higher than that of group E (P0.05). Compared with group E, the total cholesterol content in group A BX C was higher than that in group E, but there was no significant difference between group E and group P0.05.D in the detection of protein and the total content of intracellular cholesterol in the supernatant of P0.05.D group. Conclusion: the shear stress of superior sagittal sinus was increased and the expression of AIBP was decreased in dural vascular endothelium 3 months after the establishment of 1.SD rat model of venous hypertension. Under high shear stress, the expression of AIBP in cultured human umbilical vein endothelial cells was down-regulated, total cholesterol was increased, and VEGF expression was up-regulated. The results suggest that shear stress may affect angiogenesis by regulating cholesterol metabolism and VEGF/VEGFR channel.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743
本文編號：2334880
[Abstract]:Background: dural arteriovenous fistula (dural arteriovenous fistula,DAVF) is an abnormal short circuit between the artery on the dura mater and its appendages (falx cerebelli, tentorium cerebellum) and the cortical vein or venous sinus. Persistent venous hypertension can lead to cerebral hemorrhage, cerebral edema, venous infarction, cognitive impairment and other neurological disorders. In clinic, complex DAVF is still difficult to be completely cured by surgical treatment. In the study of the etiology of DAVF, it is found that angiogenesis caused by venous hypertension plays a key role. Therefore, it is important to clarify the role of angiogenesis in the pathogenesis of dural arteriovenous fistula and to control the progress of this disease. Objective: 1. A rat model with different venous pressure gradient was established to explore the effect of venous pressure change on the shear stress of superior sagittal sinus. 2. The expression of related proteins in AIBP-VEGF mediated angiogenesis pathway and in the supernatant of human umbilical vein endothelial cells under different shear stress was detected in the dura mater of rats with different venous hypertension. To explore the effect of shear stress changes caused by venous hypertension on angiogenesis regulated by AIBP. Experimental method: 1. Part one: the rat models with different venous pressure gradient were established. 80 SD rats were randomly divided into 4 groups (20 rats in each group). Group A (20 rats) underwent right common carotid jugular vein anastomosis (right common carotid jugular vein anastomosis) and left transverse sinus outlet occlusion. The right common carotid artery external jugular vein anastomosis was performed in 20 rats in group B, the right external jugular vein ligation and the right common carotid artery ligation were performed in 20 rats in group C. In group D, 20 rats were sham-operated. All rats underwent parietal bone removal after operation, exposed the superior sagittal sinus, measured and calculated the shear stress of the superior sagittal sinus 3 months later, killed the rats, took the dural tissue and detected the expression of AIBP,VEGF. 2. The second part: human umbilical vein endothelial cells were divided into four groups according to shear stress. According to the results of shear stress of the superior sagittal sinus of rats in the first part, human umbilical vein endothelial cells were cultured under the shear stress of 10 ~ (5) ~ 2 ~ (2.5) ~ (10) dyn / cm ~ (2) for 24 hours, respectively. The expression of AIBP and VEGF were detected in the supernatant of the cells and the total cholesterol content in the cells was measured. The experiment was repeated three times in each group. Experimental results: 1. Part one: the average shear stress of superior sagittal sinus in group A was 5.32 鹵0.31 dyn / cm ~ (-2) in group B, and the mean value was 2.39 鹵0.03 dyn / cm ~ (2) in group C, and 0.97 鹵0.01 dyn / cm ~ (-2) in group C, respectively. The average shear stress of superior sagittal sinus in group D was 0.88 鹵0.01dyn / cm ~ 2 路m ~ (-2). The shear stress in group B was higher than that in group D (P < 0.05), but there was no significant difference between group C and D (. Western blot and real-time PCR). The expression of AIBP and VEGF in the dura mater of group B was significantly lower than that in group D, but there was no significant difference between group D and group C. Immunohistochemical results showed that AIBP was expressed in the whole vascular layer. The second part: in the supernatant of umbilical vein endothelial cells cultured under high shear stress, the expression of AIBP was lower than that of group E, and the expression of VEGF was higher than that of group E (P0.05). Compared with group E, the total cholesterol content in group A BX C was higher than that in group E, but there was no significant difference between group E and group P0.05.D in the detection of protein and the total content of intracellular cholesterol in the supernatant of P0.05.D group. Conclusion: the shear stress of superior sagittal sinus was increased and the expression of AIBP was decreased in dural vascular endothelium 3 months after the establishment of 1.SD rat model of venous hypertension. Under high shear stress, the expression of AIBP in cultured human umbilical vein endothelial cells was down-regulated, total cholesterol was increased, and VEGF expression was up-regulated. The results suggest that shear stress may affect angiogenesis by regulating cholesterol metabolism and VEGF/VEGFR channel.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743
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