尿毒症毒素激活NLRP3炎症体信号通路介导血管新生内膜增生机制的实验研究

发布时间：2018-03-08 19:38

本文选题：慢性肾病　切入点：动静脉瘘　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.通过构建小鼠慢性肾脏病(CKD)模型和动静脉瘘(AVF)模型,探究CKD是否通过NLRP3炎症体信号通路介导的炎症反应促使血管新生内膜增生(NH)。2.明确NLPP3基因敲除对CKD小鼠血管新生内膜增生具有减缓作用,并探究NLRP3抑制剂格列本脲是否对CKD相关的血管新生内膜增生具有治疗作用。方法:1.实验分组:取健康雄性C57小鼠24只,随机分成CKD+AVF手术组、CKD伪手术组、格列本脲组及药物对照组;NLRP3基因敲除雄性C57小鼠6只为NLRP3基因敲除组。采用5/6肾切除术构建小鼠CKD模型,CKD术后1周留取小鼠血液标本。采用小鼠右侧颈总动脉与颈内静脉端端吻合术构建小鼠AVF模型,术后3周留取小鼠血管标本。2.采用苦味酸法测定小鼠血肌酐(SCr)水平,脲酶法测定血尿素氮(BUN)含量,生化法测定血管组织Caspase-1活性。3.HE染色观察血管组织病理变化,测量并分析各组小鼠新生血管内膜增生情况。4.免疫荧光双标记法分析各组血管组织NLRP3、α-SMA及NLRP3下游信号分子IL-1β与IL-18在血管新生内膜的表达水平。5.RT-PCR法分析各组血管组织NRLP3 m RNA的表达。结果:1.与CKD伪手术组相比,CKD+AVF手术组、格列本脲组、药物对照组及NLRP3基因敲除组小鼠血清SCr与BUN水平显著增高(p0.05)。2.血管组织HE染色示,与CKD伪手术组相比,CKD+AVF手术组小鼠血管内膜明显增厚,血管平滑肌细胞增生并向内膜迁移(p0.05),而格列本脲组及NLRP3基因敲除组血管内膜厚度无明显变化(p0.05)。3.与CKD伪手术组相比,CKD+AVF手术组小鼠血管组织NLRP3 m RNA表达量明显增高,Caspase-1活性显著升高,血管平滑肌细胞NLRP3、IL-1β及IL-18蛋白含量也显著增高;而格列本脲治疗组、NLRP3基因敲除组NLRP3 m RNA的表达量、Caspase-1活性及血管平滑肌细胞NLRP3、IL-1β、IL-18蛋白的含量等指标与CKD伪手术组相比无统计学差异(p0.05)。4.上述各项指标在CKD+AVF手术组与药物对照组之间的差异无统计学意义(p0.05)。结论:1.CKD产生的尿毒症毒素可诱导动静脉瘘处血管平滑肌细胞表达多种炎症细胞因子,如Caspase-1、IL-1β、IL-18,诱发血管平滑肌细胞增生并向血管内膜迁移,造成动静脉瘘处管腔狭窄。2.NLRP3基因敲除可显著抑制NLRP3炎症体信号通路下游Caspase-1、IL-1β、IL-18等炎症因子的产生,对CKD相关的血管新生内膜增生具有抑制作用。3.格列本脲抑制NLRP3炎症体信号通路激活、降低下游炎症因子Caspase-1、IL-1β和IL-18的表达,进而阻止血管平滑肌细胞增生并向血管内膜迁移有关,对CKD相关的动静脉瘘管腔狭窄的形成具有抑制作用。
[Abstract]:Objective: through the construction of 1. mice with chronic kidney disease (CKD) model and arteriovenous fistula (AVF) model, to explore whether CKD inflammation through NLRP3 body inflammation mediated signaling pathway promotes neointimal hyperplasia (NH).2. specific NLPP3 gene knockout has protective effect on neointimal hyperplasia in CKD mice and explore the NLRP3 vessels. The inhibitor glibenclamide is neointimal hyperplasia of CKD related with therapeutic effect. Methods: 1. experimental groups: 24 healthy male C57 mice were randomly divided into CKD+AVF operation group, CKD sham group, glibenclamide group and drug control group; NLRP3 gene knockout of 6 male C57 mice with NLRP3 gene knockout group. Construction of a CKD model by 5/6 nephrectomy, the mice blood specimens CKD 1 weeks after operation. The right carotid artery and internal jugular vein in mice anastomosis construct mouse AVF model, the mice blood vessel standard left after 3 weeks The.2. method for the determination of mice with picric acid (SCr) level of serum creatinine, blood urea nitrogen determination method of urease (BUN) content, determination of staining to observe the pathological changes of vascular tissue Caspase-1 activity.3.HE vascular tissue biochemical method, analysis of mice neointimal hyperplasia.4. immunofluorescence double labeling analysis of vascular tissue were NLRP3 and alpha -SMA measurement. NLRP3 and the downstream signal molecule IL-1 beta and IL-18 expression level in.5.RT-PCR analysis of expression of vascular neointimal tissue were NRLP3 m RNA. Results: 1. CKD compared with the sham operation group, CKD+AVF group, lattice glyburide group, drug control group and NLRP3 knockout mice serum SCr and BUN levels increased significantly (P0.05).2. vascular tissue HE staining showed that CKD compared with the sham operation group, CKD+AVF group of mice with vascular intima was obviously thickened, vascular smooth muscle cell proliferation and migration to intima and glibenclamide (P0.05). Urea group and NLRP3 knockout group intimal thickness had no significant change (P0.05) CKD.3. compared with the sham operation group, significantly increased the expression of RNA NLRP3 m CKD+AVF group of mice with vascular tissue, the activity of Caspase-1 was significantly increased in vascular smooth muscle cells NLRP3, IL-1 beta and IL-18 protein content was significantly increased; and glibenclamide the treatment group, the expression of NLRP3 in group M RNA NLRP3 NLRP3 Caspase-1, gene knockout, activity of vascular smooth muscle cell and IL-1 beta protein content, index of IL-18 and CKD in sham group had no statistical difference (P0.05) there was no significant difference in the above indexes between.4. CKD+AVF surgery group and drug control group (P0.05). Conclusion: 1.CKD can induce the generation of uremic toxins arteriovenous fistula vascular smooth muscle cell factor expression, inflammatory cells such as Caspase-1, IL-1 beta, IL-18, induced vascular smooth muscle cell proliferation and intimal Migration caused by arteriovenous fistula stenosis at the.2.NLRP3 gene knockout could significantly inhibit NLRP3 body inflammatory signaling pathways downstream of Caspase-1, IL-1 beta, IL-18 and other inflammatory factors, neointimal hyperplasia of CKD can inhibit the.3. glibenclamide inhibition of NLRP3 signal pathway activation of inflammation, reduce inflammatory cytokines Caspase-1 expression. IL-1 beta and IL-18, and inhibit the proliferation of vascular smooth muscle cells and intimal migration related to CKD related arteriovenous fistula can inhibit the formation of luminal stenosis.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

【相似文献】