【摘要】：研究背景和目的急性肢體缺血是一種危險性極高的動脈性疾病,發(fā)生后需緊急重建血運,恢復血流灌注,但血運恢復后會導致缺血再灌注損傷,不僅可引起局部組織損傷,還可導致遠位器官的損傷,出現(xiàn)多器官功能衰竭。TLRs通過LRRs結(jié)構(gòu)域識別病原體相關(guān)分子模式,進而刺激炎癥因子的生成,參與病原體的殺傷。TLR4受體作為TLRs的一員,在體內(nèi)廣泛分布,幾乎分布于所有的細胞系,主要表達于宿主的防御功能細胞,介導多種疾病,以炎癥反應(yīng)為主,誘導多種炎癥因子基因表達。TLR4在多種器官缺血再灌注損傷中有重要作用,如肝、腎、脊髓、大腦等器官,可以啟動免疫反應(yīng)和引發(fā)炎性反應(yīng),敲除TLR4基因后可明顯減輕缺血再灌注損傷。但TLR4在缺血再灌注血管損傷中的作用尚不清楚,本實驗擬證明TLR4在小鼠下肢缺血再灌注血管損傷中的作用。研究內(nèi)容1.應(yīng)用激光多普勒血流成像技術(shù)對小鼠下肢急性缺血再灌注模型的評估及血流動力學研究。2.野生(WT)模型組中股動脈的炎癥損傷和凋亡。3.TLR4基因敲除(TLR4-/-)模型組股動脈的炎癥損傷和凋亡。研究方法1.血流動力學實驗:本研究采用橡皮圈繞扎法建立小鼠下肢缺血再灌注模型,將C57BL/6J小鼠隨機分為模型對照組(control)、缺血組(Ischemia)和再灌注模型組(Reperfision),激光多普勒血流成像儀分別檢測各組血流灌注情況。2.缺血再灌注損傷實驗:將C57BL/6J小鼠隨機分為模型對照組(control)和缺血再灌注組(IR),免疫組化檢測TLR4、HMGB1、TNF-α及IL-6的表達,RT-PCR檢測TNF-α、IL-6的表達,TUNEL技術(shù)檢測血管壁細胞凋亡情況。3.TLR4實驗:將小鼠缺血再灌注模型分為野生(WT)模型組和TLR4基因敲除(TLR4--)模型組,RT-PCR檢測TNF-α、IL-6的表達,TUNEL技術(shù)檢測血管壁細胞凋亡情況。研究結(jié)果1.采用橡皮圈繞扎法成功建立小鼠下肢缺血再灌注模型,橡皮圈套扎后術(shù)側(cè)后爪皮膚立即蒼白,溫度降低,Flux迅速下降,健側(cè)肢體后爪未出現(xiàn)以上表現(xiàn)。再灌注后術(shù)側(cè)后爪出現(xiàn)腫脹,皮溫升高,Flux逐漸升高。2.缺血再灌注損傷實驗中,IR組血管組織的TLR4、HMGB1、TNF-α和IL-6的表達及凋亡情況較control組明顯升高(P0.01)。3.TLR4基因敲除后,TLR4 IR組管壁TNF-α和IL-6的表達減少,凋亡明顯減輕(P0.01)。結(jié)論1.采用橡皮圈繞扎法可以成功建立小鼠下肢缺血再灌注模型,肢體結(jié)扎后股動脈呈低灌注狀態(tài),再灌注后血流灌注逐漸升高。2.急性下肢缺血再灌注可導致血管組織發(fā)生炎癥反應(yīng)和凋亡。3.TLR4受體在小鼠下肢缺血再灌注損傷模型中會促進內(nèi)皮細胞凋亡和炎癥損傷。
[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- 偽 and IL-6. RT-PCR was used to detect the expression of TNF- 偽 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- 偽 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- 偽 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- 偽 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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R543

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