髓系特异性CXCR4敲除影响小鼠下肢缺血恢复的研究

发布时间：2019-05-17 08:54

【摘要】：研究背景外周血管病严重危害人类健康,而如何解决缺血发生后组织修复和功能恢复是目前的研究热点。炎症是缺血后组织修复以及血管新生不可或缺的条件,适度的炎症反应有助于清除坏死组织,促进血管新生,而过度的炎症反应可加重组织损伤,促进缺血病灶进一步恶化。趋化因子在炎症细胞浸润的过程中起到至关重要的作用。已证实多种趋化因子及其受体参与调控缺血后炎症细胞的募集,其中包括基质衍生因子(SDF-1)及其配体CXCR4.并且已有初步研究显示SDF-1/CXCR4轴参与调控缺血后炎症反应以及血管新生。然而,SDF-1/CXCR4轴在缺血相关炎症及修复中的作用仍不明了。实验目的研究CXCR4如何调控中性粒细胞以及巨噬细胞在缺血下肢处的募集,并探讨CXCR4在炎症细胞的表达对缺血性炎症反应以及修复的影响。实验方法：我们通过Cre-LoxP基因敲除技术得到了髓系细胞(主要是中性粒细胞和巨噬细胞)特异性CXCR4敲除的小鼠(MKO)。我们检测了MKO小鼠体重,脾脏大小,外周血象等指标,并检测了中性粒细胞和巨噬细胞表面CXCR4的表达。通过结扎股动脉我们构建了小鼠下肢缺血模型,通过激光多普勒观察术后小鼠下肢血流恢复的情况并观察足趾坏死情况。通过免疫荧光染色我们观察比较MKO小鼠和对照小鼠缺血组织血管新生的情况,并通过组织化学染色和流式细胞术观察炎症细胞在缺血病灶的浸润情况,并通过PCR检测缺血下肢炎症因子表达情况。在体外,我们观察SDF-1/CXCR4轴对中性粒细胞,巨噬细胞迁移能力的影响,以及观察CXCR4敲除对上述两种细胞促炎能力的影响。为进一步明确CXCR4对巨噬细胞极化的影响,我们分别通过脂多糖(LPS)和干扰素-γ以及白介素-4分别诱导MKO或WT来源的骨髓巨噬细胞向M1型和M2型极化,通过qPCR检测相关极化标志物的表达水平。实验结果我们发现MKO小鼠中性粒细胞骨髓中失归巢,在外周血数量明显增加。对照组小鼠较MKO小鼠下肢缺血恢复明显变差,足趾坏死程度更为严重,然而并不影响其远期预后。提示髓系细胞CXCR4表达对于下肢缺血血流恢复的重要作用。通过免疫荧光染色和流式细胞术我们发现较对照小鼠相比,MKO小鼠术后早期缺血肌肉CDllb阳性细胞和中性粒细胞数量明显增加,随后迅速下降,同时炎症因子的表达也有所增加。然而巨噬细胞数量并无明显改变。在体外试验,我们发现CXCR4敲除对于中性粒细胞炎症因子释放能力并无影响,明显增强了巨噬细胞由M0向M1极化的能力儿不改变其向M2极化的能力。同时CXCR4敲除并不影响了中性粒细胞对于SDF-1的迁移能力,而使巨噬细胞迁移能力受损。这些结果表明髓系细胞SDF-1/CXCR4轴敲除可增强了缺血早中期修复相的炎性反应,而对中后期修复相并无明显影响。实验结论CXCR4是调节下肢缺血后炎症细胞募集的关键趋化因子受体,CXCR4缺失可引起急性期过多的中性粒细胞细胞浸润；同时可通过促进巨噬细胞向M1极化从而加重炎症反应,然后早期增强的炎症反应反而增强了缺血早期的血流灌注恢复,提示炎症在缺血后组织修复中的作用。
[Abstract]:The study of the background of the background of the background is a serious threat to human health, and how to solve the post-ischemic tissue repair and function recovery is the current research hotspot. Inflammation is an essential condition for post-ischemic tissue repair and angiogenesis, and moderate inflammatory response can help to remove necrotic tissue, promote angiogenesis, and excessive inflammatory reaction can aggravate tissue damage and promote further deterioration of ischemic lesions. Chemokines play a critical role in the process of inflammatory cell infiltration. Various chemokines and their receptors have been shown to be involved in the control of the recruitment of post-ischemic inflammatory cells, including the matrix-derived factor (SDF-1) and its ligand CXCR4. And the preliminary studies have shown that the SDF-1/ CXCR4 axis is involved in the regulation of post-ischemic inflammatory response and angiogenesis. However, the role of the SDF-1/ CXCR4 axis in ischemia-related inflammation and repair is still unknown. Objective To study how CXCR4 regulates the recruitment of neutrophils and macrophages at the lower extremity of ischemia, and discusses the effect of CXCR4 on the inflammatory response and repair of inflammatory cells. Methods: We obtained the mouse (MKO) specific to the specific CXCR4 knock-out of the myeloid cells (mainly neutrophils and macrophages) through the Cre-LoxP gene knockout technique. We tested the weight, spleen size and peripheral blood image of MKO mice, and detected the expression of CXCR4 on the surface of neutrophils and macrophages. The lower limb ischemia model of the mice was constructed by ligation of the femoral artery, and the blood flow recovery in the lower extremities of the mice was observed by laser Doppler and the condition of the foot-toe necrosis was observed. In this paper, the expression of the inflammatory cytokines in the ischemic myocardium was detected by immunohistochemistry and flow cytometry, and the expression of the inflammatory factors of the lower limb was detected by PCR. In vitro, we observed the effects of the SDF-1/ CXCR4 axis on the ability of neutrophils and macrophages to migrate and to observe the effects of CXCR4 knockout on the two cell proinflammatory capabilities. In order to further clarify the effects of CXCR4 on the polarization of macrophages, we respectively induced the M-and M2-type polarization of bone marrow macrophages from the source of MKO or WT by lipopolysaccharide (LPS) and interferon-1 and interleukin-4, respectively, and the expression level of the related polarized markers was detected by qPCR. The results showed that the number of peripheral blood in the bone marrow of the MKO mice was significantly increased. In the control group, the lower limb ischemic recovery of the MKP mice was significantly worse, and the degree of toe necrosis was more serious, however, the long-term prognosis of the mice was not affected. It is suggested that the expression of CXCR4 in the myeloid cells plays an important role in the recovery of ischemic blood flow in the lower limbs. Compared with control mice by immunofluorescence staining and flow cytometry, the number of CDl1b positive cells and the number of neutrophils in the early ischemic muscle of the MKO mice increased significantly, and then decreased rapidly, and the expression of the inflammatory factors increased. However, that numb of macrophages did not change significantly. In vitro, we found that CXCR4 knock-out has no effect on the ability to release a neutrophil inflammatory factor, significantly enhancing the ability of macrophages to polarize M1 from M0 to M2 without changing its ability to polarize M2. At the same time, CXCR4 knock-out does not affect the ability of neutrophils to migrate to SDF-1, and the ability to migrate macrophages is impaired. These results suggest that the knock-out of the SDF-1/ CXCR4 shaft of the myeloid cells can enhance the inflammatory reaction of the early-stage repair of the ischemia, and has no significant effect on the phase of the middle and later stage. The results show that CXCR4 is a key chemokine receptor for regulating the recruitment of inflammatory cells after lower limb ischemia. The deletion of CXCR4 can cause excessive neutrophil infiltration in the acute stage, and can increase the inflammatory response by promoting the polarization of the macrophage to the M1. Then the early-enhanced inflammatory reaction enhances the early blood flow perfusion recovery in the early stage of the ischemia, and prompts the inflammation to function in the post-ischemia tissue repair.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543

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