紊流对动静脉内瘘血管内皮细胞的影响

发布时间：2018-03-26 00:37

本文选题：紊流　切入点：震荡剪切力　出处：《天津医科大学》2017年硕士论文

【摘要】：目的:动静脉内瘘(Arteriovenous fistulae,AVF)是维持性血液透析(Maintenance hemodialysis,MHD)患者的生命线,AVF吻合口处的静脉段狭窄是内瘘失功的主要原因,其病理机制为内膜增生,与特殊部位的壁面剪切力及血流模式改变有关。且透析患者体内炎症介质与氧化应激密切相关,两者之间相互作用,共同损伤血管内皮细胞。有研究表明紊流促进血管内皮细胞增殖。小窝蛋白-1(caveolin-1,Cav-1)大量存在于血管内皮细胞中,是分布于其表面的力学感受器,参与细胞内多种信号通路的调节,内皮细胞受到剪切力作用时,膜表面蛋白表达及分布会发生改变。转化生长因子-β1(transforming growth factor-β1,TGF-β1)不仅具有诱导细胞增殖和迁移的作用,还能促进细胞外基质积聚。本研究旨在研究体外生理环境中以及尿素症环境中紊流作用下Cav-1、TGF-β1在血管内皮的分布及功能的表达情况。方法:应用体外洄流模型模拟体内紊流状态,分别用平行平板流动小室来模拟血管管腔,蠕动泵来控制循环流量,注射泵制造洄流血流,含血清的培养基模拟血液,以脐静脉内皮细胞(HUVEC)模拟静脉血管内皮细胞,尿毒症患者血清按比例加入到培养基中模拟尿毒症环境。洄流作用不同时间(分别为0小时、2小时、6小时和12小时)对内皮细胞的影响。以及用低于生理水平的层流剪切力(6dynes/cm2)、生理水平的层流剪切力(12dynes/cm2)和超生理水平的层流剪切力(20dynes/cm2)以及洄流产生的震荡剪切力(0±5dynes/cm2)分别作用于HUVEC 12h,对照组不受剪切力作用。应用Western blotting和免疫荧光技术检测洄流产生的震荡剪切力作用不同时间以及不同形式的剪切力作用下Cav-1、TGF-β1的分布及功能的表达情况。结果:在洄流产生的震荡剪切力作用下,Cav-1的表达随时间的延长呈逐渐下降趋势,且其分布表现为由质膜向胞质转移。而TGF-β1的表达则恰恰相反。对比洄流和不同大小的层流剪切力作用12h,发现Cav-1在洄流产生的震荡剪切力作用下以及低于生理水平的层流剪切力作用下表达下降。Cav-1在高于生理水平的层流剪切力作用下其表达增多。而TGF-β1在洄流作用和低于生理水平的层流剪切力表达增多。在高于生理水平的层流剪切力作用下其表达有所下降。在尿毒症环境中,内皮细胞的活性比正常血清环境下有所减低。各组差异均具有统计学意义。结论:血流形式影响细胞因子的表达。紊流、低于生理水平的层流剪切力和长时间的作用使内皮细胞的结构和功能发生改变,在动静脉内瘘失功中,起到关键性作用。尿毒症环境影响血管内皮细胞中的细胞因子的表达。
[Abstract]:Objective: Arteriovenous fistulaeus AVFis is the main cause of arteriovenous fistula failure in maintenance hemodialysis patients with maintenance hemodialysis and maintenance maintenance hemodialysis (MHD), and its pathological mechanism is intimal hyperplasia. And the inflammatory mediators in dialysis patients are closely related to oxidative stress, and they interact with each other. Some studies have shown that turbulence promotes the proliferation of vascular endothelial cells. Fossa protein -1caveolin-1 (Cav-1) is abundant in vascular endothelial cells and is a mechanical receptor distributed on its surface, which is involved in the regulation of various signal pathways in cells. When endothelial cells were subjected to shear stress, the expression and distribution of membrane proteins changed. Transforming growth factor-尾 1(transforming growth factor- 尾 1 (TGF- 尾 1) could not only induce cell proliferation and migration, but also induce cell proliferation and migration. The aim of this study was to study the distribution and function of Cav-1 TGF- 尾 1 in vascular endothelium in vitro and in uremic environment. Parallel flat flow chamber was used to simulate vascular lumen, peristaltic pump to control circulation flow, injection pump to make blood flow, serum-containing medium to simulate blood, and umbilical vein endothelial cell (HUVECs) to simulate venous endothelial cells. The serum of uremic patients was proportionally added to the medium to simulate the uremic environment. The effects of whirl on endothelial cells at different times (0 hours / 2 hours / 6 hours and 12 hours respectively). Laminar shear stress (6dynes / cm2), physiological laminar shear stress (12dynes / cm2), superphysiological laminar shear force (20dynes / cm2) and whirlwind shear force (0 卤5dynes / cm2) were applied to HUVEC for 12 h, respectively. The control group was not subjected to shear stress. Western blotting and immunofluorescence were used. The distribution of Cav-1 TGF- 尾 1 and the expression of TGF- 尾 1 under different time and different forms of shear force were detected by the technique. Results: the expression of Cav-1 was expressed over time under the shear force generated by the whirl. The prolongation showed a decreasing trend. The expression of TGF- 尾 1 was the opposite. Compared with the swirl current and laminar shear force of different sizes for 12 h, it was found that Cav-1 was subjected to oscillatory shear force generated by whirl flow and was lower than physiological level. The expression of Cav-1 increased under laminar shear stress, while TGF- 尾 1 increased under whirl flow and laminar shear stress below physiological level, while TGF- 尾 1 increased under laminar shear stress above physiological level. The expression of fluid shear stress decreased. In uremic environment, The activity of endothelial cells was lower than that of normal serum environment. The differences were statistically significant. Conclusion: the form of blood flow affects the expression of cytokines. Laminar shear stress at lower than physiological level and long-term action changed the structure and function of endothelial cells and played a key role in arteriovenous fistula dysfunction. Uremia environment affected the expression of cytokines in vascular endothelial cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

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