阿托伐他汀对静脉桥的保护作用及机制初探

发布时间：2018-05-11 01:05

本文选题：冠状动脉旁路移植术 + 静脉桥　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:冠状动脉旁路移植术(coronary artery bypass graft,CABG)作为一种稳定而有效的血管重建方法[1]被广泛地应用于治疗冠状动脉粥样硬化性心脏病,但其远期疗效不甚理想。血管内膜增生是导致血管重建术后自体静脉桥管腔狭窄或闭塞的主要原因,已被大量的研究所证实。引起静脉移植再狭窄的主要原因是血管内皮损伤,其主要是由于血管内膜增生,血管平滑肌细胞(vascular smooth muscle,VSMCs)过度增殖及分泌大量细胞外基质。即再狭窄是一种由于手术的物理性损伤导致的缺血再灌注损伤后的局部血管修复反应,是血管重塑的过程。因此,阻断血管平滑肌的过度增殖是预防CABG术后静脉桥再狭窄的有效策略。大量的研究证实,阿托伐他汀能够抑制血管平滑肌的增殖,据此推测,阿托伐汀可能阻断CABG术后发生静脉桥再狭窄,但其发生机制至今尚未阐明。NF-κB是从B淋巴细胞核中检测到的一种能与免疫球蛋к轻链基因的增强子κB序列特异结合的核蛋白因子。非活化状态下NF-κB以与IκB聚合的三聚体形式或与前体蛋白聚合的二聚体形式存在于胞浆中,在多种细胞因子的作用下,通过多种信号途径IκB发生磷酸化并与NF-κB解聚,NF-κB被激活,将信息由胞浆传递至胞核并启动相关靶基因的转录,参与自由基损伤、细胞凋亡、免疫调节、炎症反应等病理生理过程[2]。此外,还有一些研究发现,当血管内皮细胞损伤发生时,多种炎症介质和细胞因子释放,激活NF-κB,NF-κB二聚体被活化,NF-κB激活后上调平滑肌细胞及内皮细胞等基因表达,引起血管内膜增生而诱导管腔狭窄。但CABG术后静脉桥血管内皮细胞增殖是否通过NF-κB信号转导途径诱导再狭窄,尚有待阐明。为此,本实验旨在观察CABG术后静脉桥血管内皮细胞增殖是否通过NF-κB信号转导通路诱导再狭窄,探讨阿托伐他汀是否通过NF-κB信号通路发挥对静脉桥的保护作用。方法:健康雄性新西兰大白兔(3±0.5Kg)40只,建立自体静脉移植动物模型。1阿托伐他汀钙对静脉桥血管壁内膜厚度、平滑肌细胞数及超微结构的影响。具体分组如下:①假手术组(sham)组(n=10):仅分离左侧颈外静脉,不行血管移植,假手术1d起,经胃管灌注生理盐水;②移植对照组(control)组(n=10):静脉移植术后1d起,经胃管灌注生理盐水;③阿托伐他汀钙(atorvastatin)5mg组(n=10):静脉移植术后1d起,经胃管灌注阿托伐他汀,剂量为5mg/kg.d;④阿托伐他汀钙(atorvastatin)10mg组(n=10):静脉移植术后1d起,经胃管灌注阿托伐他汀,剂量为10mg/kg.d。每组共处理4w,在静脉移植后或假手术后4w取静脉桥,分别用HE染色法、免疫组化、电镜观察阿托伐他汀对静脉桥血管壁内膜厚度、平滑肌细胞数及超微结构的影响。2阿托伐他汀钙对NF-κB蛋白及NF-κB p50/p65二聚体复合物表达的影响。具体分组如下:①假手术组(sham)组(n=10):仅分离左侧颈外静脉,不行血管移植,假手术1d起,经胃管灌注生理盐水;②移植对照组(control)组(n=10):静脉移植术后1d起,经胃管灌注生理盐水;③阿托伐他汀钙(atorvastatin)5mg组(n=10):静脉移植术后1d起,经胃管灌注阿托伐他汀,剂量为5mg/kg.d;④阿托伐他汀钙(atorvastatin)10mg组(n=10):静脉移植术后1d起,经胃管灌注阿托伐他汀,剂量为10mg/kg.d。每组共处理4w,在静脉移植术后或假手术后4w取静脉桥,应用Western blot方法观察NF-κB蛋白表达的变化,免疫共沉淀(Co-immunoprecipitation)方法观察NF-κB p50/p65二聚体复合物表达的变化。探讨阿托伐他汀对NF-κB表达的影响。结果:1阿托伐他汀钙对静脉桥血管壁内膜厚度的影响采用HE染色方法,在光镜下观察静脉桥移植术后,静脉桥血管壁内膜厚度的变化。Control组(Fig.2)、atoravastatin 5mg组(Fig.3)、atoravastatin 10mg组(Fig.4)静脉桥血管管壁与sham组(Fig.1)相比,均有不同程度的增厚(P0.05)。与Sham组(Fig.1)相比,Control组(Fig.2)静脉桥血管壁明显增厚(P0.01)。与Control组(Fig.2)相比,atoravastatin 5 mg组(Fig.3)无显著性差异(P0.05);atoravastatin 10 mg组(Fig.4)静脉桥血管管壁平滑,内膜增生程度明显减少(P0.05)。2阿托伐他汀钙对静脉桥血管壁内膜平滑肌数目的影响采用免疫组化方法,在光镜下观察静脉桥移植术后,静脉桥血管壁内膜平滑肌细胞数目的变化。Control组(Fig.6)、atoravastatin 5 mg组(Fig.7)、atoravastatin 10 mg组(Fig.8)静脉桥血管内皮及平滑肌层与sham组(Fig.5)相比,均有不同程度的增殖(P0.05)。与Sham组(Fig.5)相比,Control组(Fig.6)有大量增殖细胞深染(P0.01);atoravastatin 5 mg组(Fig.7)深染的增殖细胞虽较Control组有所减少,但无明显变化(P0.05);atoravastatin 10 mg组(Fig.8)深染的增殖细胞明显减少(P0.05)。3阿托伐他汀钙对静脉桥血管壁超微结构的影响在电镜下观察静脉桥移植术后,静脉桥血管壁超微结构的变化。Control组(Fig.9)可见静脉桥血管壁内膜明显增厚。与Control组(Fig.9)相比,atoravastatin 5 mg组(Fig.10),静脉桥血管壁内膜有一定程度的增厚,虽较Control组(Fig.9)少,但无显著差异(P0.05);atoravastatin 10 mg组(Fig.11)内膜连续性好(P0.05)。4阿托伐他汀钙对NF-κB蛋白表达的影响应用Western blot方法观察静脉桥移植术后,阿托伐他汀钙对NF-κB蛋白表达的影响NF-κB p50(Fig.12)在Sham组、control组、atoravastatin 5 mg组、atoravastatin 10 mg均有一定的表达量。Sham表达量较少。与Sham组相比,control组、atoravastatin 5 mg组、atoravastatin 10 mg组其蛋白表达均有所上调(P0.05)。与control组相比,atoravastatin 5 mg组其蛋白表达虽有所下调,但无显著性差异(P0.05),atoravastatin 10 mg组此蛋白表达明显下调(P0.05)。上述结果表明,阿托伐他汀钙能够显著下调NF-κB p50的表达。5阿托伐他汀钙对NF-κB p50/p65二聚体复合物表达的影响通过免疫共沉淀(Co-immunoprecipitation)的方法研究了静脉桥移植术后,阿托伐他汀钙对NF-κB p50/p65二聚体复合物表达的影响。NF-κB p50/p65二聚体复合物(Fig.12)在Sham组、control组、atoravastatin 5 mg组、atoravastatin 10 mg组均有表达,其中control组此二聚体复合物表达最多。与Sham组相比,control组其表达显著上调(P0.01);atoravastatin 5 mg组此二聚体复合物表达虽有所下调,但明显高于Sham组(P0.05)。与control组相比,atoravastatin 10 mg组其表达明显下调,差异有显著性(P0.01)。上述结果表明,阿托伐他汀钙能够有效阻断静脉桥移植术后NF-κB p50/p65二聚体复合物数量的上调。小结:1静脉桥移植术后,NF-κB的蛋白表达显著上调;NF-κB被激活,即表现为NF-κB发生二聚体化,NF-κB二聚体复合物明显上调,并由胞浆转移至胞核。2静脉桥移植术后,经10 mg atoravastatin处理,下调NF-κB p50蛋白的表达,下调NF-κB p50/p65的表达二聚体复合物的表达。结论:静脉桥移植术后,静脉桥血管内皮细胞增殖通过NF-κB信号通路诱导再狭窄;阿托伐他汀钙通过NF-κB信号通路发挥对静脉桥的保护作用。
[Abstract]:Objective: coronary artery bypass graft (CABG), as a stable and effective vascular reconstruction method, is widely used in the treatment of coronary atherosclerotic heart disease, but its long-term effect is not satisfactory. Vascular intima hyperplasia is the cause of autogenous vena cava stenosis or occlusion after the reconstruction of blood tube. The main cause has been confirmed by a large number of studies. The main cause of restenosis is vascular endothelial damage, mainly due to vascular intima hyperplasia, vascular smooth muscle cells (vascular smooth muscle, VSMCs) excessively proliferation and secretion of a large number of extracellular matrix. Restenosis is a result of physical damage caused by surgery. The local vascular repair reaction after ischemia-reperfusion injury is a process of vascular remodeling. Therefore, blocking the excessive proliferation of vascular smooth muscle is an effective strategy to prevent the restenosis after CABG. A large number of studies have confirmed that atorvastatin can inhibit the proliferation of vascular smooth muscle. Accordingly, it is presumed that opavastin may block the post operation of CABG. However, the mechanism of.NF- kappa B is a nuclear protein factor that is specific to the enhancer kappa B sequence that can be detected from the B lymphocyte nucleus. NF- kappa B is in the form of polymerization with I kappa B or in the form of two polymer polymerization with the precursor protein in the non activated state. In the cytoplasm, I kappa B is phosphorylated and depolymerization with NF- kappa B through a variety of signaling pathways, and NF- kappa B is deactivated. NF- kappa B is activated. The information is transferred from the cytoplasm to the nucleus and activates the transcription of the related target genes, and participates in the pathological and physiological processes, such as free radical damage, cell apoptosis, immunoregulation, and inflammatory reaction, [2]., and some other studies. It was found that when vascular endothelial cell injury occurred, many inflammatory mediators and cytokines were released, activated NF- kappa B, NF- kappa B two polymer was activated. After activation of NF- kappa B, the gene expression of smooth muscle cells and endothelial cells was up-regulated, causing intima hyperplasia to induce the stenosis of the lumen, but whether the proliferation of vascular endothelial cells in vein bridge after CABG has passed NF-. In this experiment, the aim of this experiment was to observe whether the proliferation of vascular endothelial cells in vein bridge after CABG was restenosis through the NF- kappa B signal transduction pathway, and to explore the protective effect of atorvastatin on the vein bridge through the NF- kappa B signaling pathway. Methods: healthy male New Zealand white rabbits (B). 3 + 0.5Kg) 40 rats, the effect of autologous vein transplantation animal model.1 atorvastatin calcium on the intima thickness, the number of smooth muscle cells and the ultrastructure of the vascular wall of the vein bridge were established as follows: (1) the sham operation group (sham) group (n=10): only the left external jugular vein, the blood tube transplantation, the false operation 1D, the saline infusion through the gastric tube, and the transplantation Control group (control) group (n=10): 1D after intravenous transplantation, saline infusion through gastric tube, and group of atorvastatin calcium (atorvastatin) 5mg (n=10) group (n=10): intravenous infusion of atorvastatin after intravenous transplantation, perfusion of atorvastatin through gastric tube, dosage of 5mg/kg.d; (n=10) atorvastatin calcium (atorvastatin) 10mg group (n=10): after vein transplantation, 1D, perfusion of atorvastatin through gastric tube The statins were treated with a dose of 10mg/kg.d., each group was treated with a total of 4W. After the vein graft or after the sham operation, the intravenous bridge was taken by 4W. HE staining, immunohistochemistry, and electron microscopy were used to observe the effect of atorvastatin on the intima thickness, the number of smooth muscle cells and the ultrastructure of the vascular wall of the vein bridge,.2 alfavastine calcium to NF- kappa B protein and NF- kappa B p50/p65 two polymer complex. The effects of the expression were as follows: (1) the sham operation group (sham) group (n=10): only the left external jugular vein, no vascular transplantation, the sham operation 1D, the saline infusion through the gastric tube, and the control group (control) group (n=10): 1D after the vein transplantation, the saline infusion through the gastric tube, and the atorvastatin calcium (atorvastatin) 5mg group (n=10): vein (n=10): vein After 1D, perfusion of atorvastatin through gastric tube, dosage of 5mg/kg.d, and atorvastatin calcium (atorvastatin) 10mg group (n=10): 1D after intravenous transplantation, perfusion of atorvastatin through gastric tube, the dose of 10mg/kg.d. in each group was treated with 4W, and the venous bridge was taken 4W after the vein graft or after the false operation, and Western blot method was used to observe NF- kappa eggs. The changes in the expression of white expression and immunoprecipitation (Co-immunoprecipitation) method were used to observe the changes in the expression of NF- kappa B p50/p65 two polymer complex. The effect of atorvastatin on the expression of NF- kappa B was investigated. Results: the effect of 1 atorvastatin calcium on the intima thickness of vascular wall intima of venous bridge was observed by HE staining, and the vein bridge transplantation was observed under the light microscope. .Control group (Fig.2), atoravastatin 5mg group (Fig.3), atoravastatin 10mg group (Fig.4), atoravastatin 10mg group (Fig.4), the vascular wall of venous bridge was thickened in varying degrees (P0.05). Compared with the Sham group, the vascular wall of the vein bridge was thickened significantly. The Vastatin 5 mg group (Fig.3) had no significant difference (P0.05), atoravastatin 10 mg group (Fig.4), the vascular wall of the vein bridge was smooth and the degree of intimal hyperplasia decreased significantly (P0.05). The effect of.2 atorvastatin calcium on the number of intima smooth muscle of vascular wall of the vein bridge was adopted by immunohistochemical method. The vascular wall of the vein bridge was observed under the light microscope. The number of intimal smooth muscle cells in group.Control (Fig.6), atoravastatin 5 mg group (Fig.7), atoravastatin 10 mg group (Fig.8), vascular endothelium and smooth muscle layer of the vein bridge were different to the sham group (P0.05). Compared with the Sham group, there were a large number of proliferating cell deep staining; 5 The proliferating cells of group Mg (Fig.7) were less than those in the Control group, but there was no significant change (P0.05). The proliferation of atoravastatin 10 mg group (Fig.8) decreased significantly (P0.05) the effect of.3 atorvastatin calcium on the ultrastructure of vein bridge wall ultrastructure under electron microscope observation of vein bridge transplantation, the changes of vascular wall ultrastructure of vein bridge In group.Control (Fig.9), the intima of vascular wall of vein bridge was obviously thickened. Compared with group Control (Fig.9), atoravastatin 5 mg group (Fig.10), the intima of vascular wall of vein bridge was thickened to a certain extent, although less than Control group (Fig.9), but there was no significant difference (P0.05). The effect of F- kappa B protein expression was used to observe the effect of Western blot method on the expression of NF- kappa B protein after vein bridge transplantation. NF- kappa B P50 (Fig.12) was in Sham group, control group and 5 groups. The protein expression of group atoravastatin 10 mg was up to up (P0.05). Compared with group control, the protein expression of group atoravastatin 5 mg decreased, but there was no significant difference (P0.05), and the expression of this protein in group atoravastatin 10 mg decreased significantly (P0.05). The above results showed that atorvastatin calcium could significantly reduce the expression of NF- kappa B. The effect of atorvastatin calcium on the expression of NF- kappa B p50/p65 two polymer complex was studied by immunoprecipitation (Co-immunoprecipitation) method to study the effect of atorvastatin calcium on the expression of NF- kappa B p50/p65 two polymer complex (.NF- kappa B p50/p65 two) complex (Fig.12) in Sham, 5 Group mg was expressed in group atoravastatin 10 mg, and the expression of this two polymer complex was the most in group control. Compared with Sham group, the expression of control group was significantly up (P0.01). The expression of this two polymer complex in atoravastatin 5 mg group was down, but obviously higher than Sham group (P0.05). Compared with control group, the expression of 10 groups was obviously expressed. Down regulation, the difference was significant (P0.01). The above results showed that atorvastatin calcium could effectively block the increase in the number of NF- kappa B p50/p65 two polymer complex after vein bridge transplantation. Conclusion: after 1 vein bridge transplantation, the protein expression of NF- kappa B was significantly up-regulated, NF- kappa B was activated, that is, NF- kappa B two polycondensation, NF- kappa B two polymer complex. After 10 mg atoravastatin treatment, the expression of NF- kappa B P50 protein was downregulated and the expression of NF- kappa B p50/p65 was down regulated by 10 mg atoravastatin treatment. Conclusion: the vascular endothelial cell proliferation of vein bridge was restenosis by NF- kappa B signaling pathway after vein bridge transplantation; atorvastatin. Calcium plays a protective role in the vein bridge through the NF- B signaling pathway.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

【参考文献】