静脉高压导致的剪应力改变在AIBP调控VEGF表达中的作用
发布时间：2018-11-16 07:37
【摘要】：研究背景:硬脑膜动静脉瘘(dural arteriovenous fistula,DAVF)是指硬脑膜及其附属物(大脑镰、小脑幕)上的动脉与脑皮层静脉或静脉窦间发生的异常血管短路,持续性的静脉高压会引发脑出血、脑水肿、静脉性梗死、认知功能下降等神经功能障碍。在临床上,复杂性DAVF仍然难以通过外科治疗得到完全治愈,明确该病发生发展的原因尤为关键。在对DAVF的病因学的研究中,发现静脉高压导致的血管新生起到关键作用,因此明确血管新生在硬脑膜动静脉瘘发病机制中的作用,成为控制该类疾病进展的重点。实验目的:1.建立不同静脉端压力梯度的大鼠模型,探索静脉端压力改变对上矢状窦剪应力的影响。2.检测AIBP-VEGF介导的血管新生通路中相关蛋白,在不同静脉高压大鼠模型硬脑膜中、在不同剪应力人脐静脉内皮细胞上清液中的表达情况,探索静脉高压造成的剪应力改变对AIBP调控血管新生的影响。实验方法:1.第一部分:建立不同静脉压力梯度的大鼠模型,将80只SD大鼠随机分为4组,每组20只,其中A组20只大鼠行右侧颈总动脉颈外静脉吻合术+左侧横窦出口闭塞术;B组20只大鼠行右侧颈总动脉颈外静脉吻合术;C组20只大鼠行右侧颈外静脉结扎术及右侧颈总动脉结扎术;D组20只大鼠为假手术组,所有大鼠于术后行顶骨磨除术,暴露上矢状窦,测量并计算3个月后上矢状窦剪应力,处死大鼠后,取硬膜组织,检测AIBP、VEGF的表达情况。2.第二部分:按剪应力不同将人脐静脉内皮细胞分为ABCDE四组,根据第一部分大鼠上矢状窦剪应力结果,分别于10、5、2.5、1、0dyn/cm2剪应力环境下培养人脐静脉内皮细胞24小时,取细胞上清行AIBP及VEGF的表达检测,取细胞检测细胞总胆固醇的含量,该部分实验每组各重复进行三次。实验结果:1.第一部分:A组大鼠上矢状窦剪应力为5.32±0.31dyn/cm2,B组大鼠上矢状窦剪应力平均值2.39±0.03dyn/cm2,C组大鼠上矢状窦剪应力平均值0.97±0.01dyn/cm2,D组大鼠上矢状窦剪应力平均值0.88±0.01dyn/cm2,A、B组剪应力高于D组,有统计学意义,C、D两组无统计学意义。Western blot及real-time PCR结果为A、B组大鼠硬脑膜中AIBP的表达明显降低,VEGF表达明显增高,与D组之间有统计学差异,C、D组无统计学意义。免疫组化结果可见AIBP在血管全层均有表达。第二部分:A、B、C组高剪应力培养下的脐静脉内皮细胞上清液中,AIBP表达量较E组降低,VEGF表达量较E组升高,P0.05;A、B、C组细胞总胆固醇含量较E组表达量升高,P0.05。D组细胞上清中检测蛋白及细胞内胆固醇总含量和E组相比,均无统计学意义。实验结论:1.SD大鼠静脉高压模型建立成功3个月后,上矢状窦的剪应力增高,硬膜血管内皮中AIBP表达下降。2.高剪应力条件下培养的人脐静脉内皮细胞AIBP的表达下调,细胞内总胆固醇增加,VEGF表达上调。提示剪应力可能通过调节胆固醇代谢,调控VEGF/VEGFR通道,影响血管新生。
[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 卤0.31 dyn / cm ~ (-2) in group B, and the mean value was 2.39 卤0.03 dyn / cm ~ (2) in group C, and 0.97 卤0.01 dyn / cm ~ (-2) in group C, respectively. The average shear stress of superior sagittal sinus in group D was 0.88 卤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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
本文编号：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 卤0.31 dyn / cm ~ (-2) in group B, and the mean value was 2.39 卤0.03 dyn / cm ~ (2) in group C, and 0.97 卤0.01 dyn / cm ~ (-2) in group C, respectively. The average shear stress of superior sagittal sinus in group D was 0.88 卤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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
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