Toll样受体4在下肢缺血再灌注血管损伤中的作用

发布时间：2018-12-13 19:04

【摘要】：研究背景和目的急性肢体缺血是一种危险性极高的动脉性疾病,发生后需紧急重建血运,恢复血流灌注,但血运恢复后会导致缺血再灌注损伤,不仅可引起局部组织损伤,还可导致远位器官的损伤,出现多器官功能衰竭。TLRs通过LRRs结构域识别病原体相关分子模式,进而刺激炎症因子的生成,参与病原体的杀伤。TLR4受体作为TLRs的一员,在体内广泛分布,几乎分布于所有的细胞系,主要表达于宿主的防御功能细胞,介导多种疾病,以炎症反应为主,诱导多种炎症因子基因表达。TLR4在多种器官缺血再灌注损伤中有重要作用,如肝、肾、脊髓、大脑等器官,可以启动免疫反应和引发炎性反应,敲除TLR4基因后可明显减轻缺血再灌注损伤。但TLR4在缺血再灌注血管损伤中的作用尚不清楚,本实验拟证明TLR4在小鼠下肢缺血再灌注血管损伤中的作用。研究内容1.应用激光多普勒血流成像技术对小鼠下肢急性缺血再灌注模型的评估及血流动力学研究。2.野生(WT)模型组中股动脉的炎症损伤和凋亡。3.TLR4基因敲除(TLR4-/-)模型组股动脉的炎症损伤和凋亡。研究方法1.血流动力学实验:本研究采用橡皮圈绕扎法建立小鼠下肢缺血再灌注模型,将C57BL/6J小鼠随机分为模型对照组(control)、缺血组(Ischemia)和再灌注模型组(Reperfision),激光多普勒血流成像仪分别检测各组血流灌注情况。2.缺血再灌注损伤实验:将C57BL/6J小鼠随机分为模型对照组(control)和缺血再灌注组(IR),免疫组化检测TLR4、HMGB1、TNF-α及IL-6的表达,RT-PCR检测TNF-α、IL-6的表达,TUNEL技术检测血管壁细胞凋亡情况。3.TLR4实验:将小鼠缺血再灌注模型分为野生(WT)模型组和TLR4基因敲除(TLR4--)模型组,RT-PCR检测TNF-α、IL-6的表达,TUNEL技术检测血管壁细胞凋亡情况。研究结果1.采用橡皮圈绕扎法成功建立小鼠下肢缺血再灌注模型,橡皮圈套扎后术侧后爪皮肤立即苍白,温度降低,Flux迅速下降,健侧肢体后爪未出现以上表现。再灌注后术侧后爪出现肿胀,皮温升高,Flux逐渐升高。2.缺血再灌注损伤实验中,IR组血管组织的TLR4、HMGB1、TNF-α和IL-6的表达及凋亡情况较control组明显升高(P0.01)。3.TLR4基因敲除后,TLR4 IR组管壁TNF-α和IL-6的表达减少,凋亡明显减轻(P0.01)。结论1.采用橡皮圈绕扎法可以成功建立小鼠下肢缺血再灌注模型,肢体结扎后股动脉呈低灌注状态,再灌注后血流灌注逐渐升高。2.急性下肢缺血再灌注可导致血管组织发生炎症反应和凋亡。3.TLR4受体在小鼠下肢缺血再灌注损伤模型中会促进内皮细胞凋亡和炎症损伤。
[Abstract]:Background and objective Acute limb ischemia is a very dangerous arterial disease. It is necessary to rebuild the blood vessel and restore the blood flow perfusion after the occurrence of acute limb ischemia. However, the recovery of blood circulation will lead to ischemia reperfusion injury, which can not only cause local tissue damage. TLRs recognizes pathogen-associated molecular patterns through the LRRs domain, which stimulates the production of inflammatory factors and participates in the killing of pathogens. TLR4 receptor is a member of TLRs. Widely distributed in the body, distributed in almost all cell lines, mainly expressed in host defense function cells, mediating a variety of diseases, mainly inflammatory response, TLR4 plays an important role in ischemia-reperfusion injury of many organs, such as liver, kidney, spinal cord, brain and other organs. Knockout of TLR4 gene significantly alleviated ischemia reperfusion injury. However, the role of TLR4 in ischemia-reperfusion vascular injury is not clear. The purpose of this experiment is to prove the role of TLR4 in ischemia reperfusion vascular injury of lower extremity in mice. Research content 1. The evaluation and hemodynamics of acute ischemia reperfusion model of lower extremity in mice were studied by laser Doppler flow imaging. 2. Inflammatory injury and apoptosis of femoral artery in wild (WT) model group and inflammatory injury and apoptosis of femoral artery in 3.TLR4 gene knockout (TLR4-/-) model group. Method 1. Hemodynamic experiment: in this study, C57BL/6J mice were randomly divided into model control group, (control), ischemia group, (Ischemia) group and reperfusion model group (Reperfision),. The blood perfusion of each group was measured by laser Doppler flow imager. 2. 2. Ischemia-reperfusion injury experiment: C57BL/6J mice were randomly divided into model control group (control) and ischemia-reperfusion group (IR),) to detect the expression of TLR4,HMGB1,TNF- 伪 and IL-6. RT-PCR was used to detect the expression of TNF- 伪 and IL-6. TUNEL technique was used to detect apoptosis of vascular parietal cells. 3.TLR4 experiment: the mice were divided into wild (WT) model group and TLR4 gene knockout (TLR4--) model group. RT-PCR was used to detect the expression of TNF- 伪 and IL-6. Apoptosis of vascular wall cells was detected by TUNEL technique. Results 1. The mouse lower extremity ischemia-reperfusion model was successfully established by rubber band ligation. After rubber band ligation, the skin of the lateral claw was immediately pale, the temperature decreased, the Flux decreased rapidly, but there was no such manifestation in the normal side of the limb. After reperfusion, the side paws were swollen, the skin temperature increased, and the Flux increased gradually. 2. The expression and apoptosis of TLR4,HMGB1,TNF- 伪 and IL-6 in vascular tissue of IR group were significantly higher than those in control group (P0.01). After 3.TLR4 gene knockout, the expression of TNF- 伪 and IL-6 in TLR4 IR group decreased. Apoptosis was significantly reduced (P0.01). Conclusion 1. The model of lower extremity ischemia reperfusion in mice was successfully established by rubber band ligation. The femoral artery showed a low perfusion state after limb ligation, and the blood flow perfusion increased gradually after reperfusion. 2. Acute lower limb ischemia-reperfusion can induce inflammation and apoptosis in vascular tissue, and 3.TLR4 receptor can promote endothelial cell apoptosis and inflammatory injury in mouse lower extremity ischemia-reperfusion injury model.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543

【相似文献】