巨噬细胞PPARγ对皮肤伤口愈合的作用研究

发布时间：2018-03-04 07:07

  本文选题：皮肤伤口愈合　切入点：巨噬细胞　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：皮肤是人体最大的器官,具有抵御冷热刺激,阻止病原体入侵等重要人体保护功能。由于皮肤常常暴露在外界环境中,极易损伤[1]。皮肤损伤后,机体启动修复维持皮肤自身稳态,恢复其完整结构和正常功能[2]。皮肤伤口愈合是一个由免疫细胞,角质上皮细胞,成纤维细胞,血管内皮细胞,细胞外基质,细胞因子以及生长因子等多细胞多介质共同参与,多阶段有序进行的复杂生理过程。可以分为组织炎症期,组织生成期和组织塑形期。正因为皮肤伤口愈合的复杂性,愈合进程极易受自身条件和外界环境的干扰而出现障碍,遗留下慢性难愈伤口。了解正常皮肤伤口愈合的调控机制可以为临床治疗慢性难愈伤口提供理论依据。巨噬细胞是调控皮肤伤口愈合的关键细胞[1,6,7]。巨噬细胞分泌促炎症因子促进伤口炎症反应,分泌蛋白酶和活性氧自由基抵抗病原体[8-10],分泌抗炎症因子和吞噬凋亡细胞促进伤口炎症消退,以及分泌细胞因子,生长因子和趋化因子等作用于角质上皮细胞,成纤维细胞和血管内皮细胞促进伤口上皮再生,胶原沉积和血管生成[11]。在伤口愈合中,巨噬细胞趋化障碍,吞噬凋亡细胞功能受损,极化异常等都将导致伤口迁延不愈[11-14]。因此,维持伤口巨噬细胞正常功能对伤口愈合至关重要。目前,对伤口巨噬细胞功能的调控机制还不清楚。核转录因子过氧化物酶体增殖物激活受体(Peroxisome proliferator activated receptorγ,PPARγ)是调控巨噬细胞功能的重要分子[15]。PPARγ可以促进巨噬细胞吞噬凋亡细胞[15,16]并使巨噬细胞从促炎症状态转化为抗炎症状态,进而促进炎症消退[17,18]。提示PPARγ在皮肤伤口愈合中对巨噬细胞可能具有一定的调控作用。我们的研究在于明确巨噬细胞PPARγ在皮肤伤口愈合中的作用及机制。在本研究中,我们首先检测了野生型(Wild type,WT)小鼠伤口愈合过程中伤口组织以及伤口巨噬细胞PPARγ的表达变化,发现皮肤损伤后组织和巨噬细胞PPARγ表达上调,提示组织和巨噬细胞PPARγ对伤口愈合具有调节作用。然后我们采用Lox P-Cre系统构建了特异敲除巨噬细胞PPARγ(PPARγ-KO)小鼠,同时将基因组含有Lox P位点但不含Cre酶(无巨噬细胞PPARγ敲除,PPARγ-WT)的小鼠作为对照。我们研究发现,PPARγ-KO小鼠伤口愈合明显延迟,伤口肉芽组织生成,胶原沉积和血管生成减少。我们进一步探究PPARγ-KO小鼠伤口愈合延迟的原因。因为炎症细胞在伤口的正常浸润对伤口愈合至关重要[19-25],我们在PPARγ-WT和PPARγ-KO小鼠伤口中检测了中性粒细胞和巨噬细胞的数目,发现没有明显差异,提示巨噬细胞PPARγ敲除不影响伤口炎症细胞浸润。除炎症细胞浸润外,炎症因子在伤口愈合中也具有重要作用。文献报道伤口TNF-α过度表达严重损害愈合过程[26-31]。我们研究发现PPARγ-KO伤口TNF-α表达明显增多,并且在PPARγ-KO伤口注射抗小鼠TNF-α拮抗剂(Anti-mouse TNF-α,a TNF-α)后伤口愈合明显加快,肉芽组织生成,胶原沉积和血管生成也明显增加。由于巨噬细胞是伤口组织细胞因子的主要来源,我们检测了PPARγ-KO伤口巨噬细胞TNF-α表达后发现其表达明显增加。这些结果说明PPARγ-KO伤口TNF-α过多表达是其愈合障碍的关键因素,并且PPARγ-KO巨噬细胞分泌的过多TNF-α促使了伤口TNF-α表达过多。我们进一步研究了PPARγ缺陷引起巨噬细胞分泌过多TNF-α的内在机制。在体外实验中我们发现,PPARγ-WT和PPARγ-KO在静息状态下都低表达TNF-α。在脂多糖(Lipopolysaccharide,LPS)刺激下,TNF-α表达明显增多但两者间没有差异,说明PPARγ对巨噬细胞分泌TNF-α没有直接调控作用。然而,在LPS刺激下加入凋亡胸腺细胞(Apoptotic thymocytes,ATs)后PPARγ-WT巨噬细胞TNF-α表达明显减少,而PPARγ-KO巨噬细胞TNF-α表达并没有减少,由于巨噬细胞吞噬凋亡细胞后TNF-α表达会减少,因此提示PPARγ-KO巨噬细胞吞噬凋亡细胞功能障碍。在LPS,ATs和细胞松弛素B(Actin-filament polymerisation-blocking agent cytochalasin B)共同孵育下,PPARγ-WT和PPARγ-KO巨噬细胞都持续高表达TNF-α,说明TNF-α表达减少确由吞噬引起,并且巨噬细胞吞噬二醋酸羧基荧光素(CFSE)标记ATs实验进一步证明了PPARγ-KO巨噬细胞吞噬率下降。在体内实验中,我们发现PPARγ-KO伤口凋亡细胞聚集,没有得到及时有效的吞噬清除。这些结果证实了PPARγ缺陷导致巨噬细胞吞噬凋亡细胞功能障碍。巨噬细胞吞噬功能与其吞噬相关受体和调理素(Opsonin)直接相关,我们检测了PPARγ-WT和PPARγ-KO巨噬细胞吞噬受体CD36,Mertk以及调理素Mfge8,Gas-6,C1qa,C1qb和C1qc的表达,结果表明PPARγ-KO巨噬细胞其吞噬相关分子表达下降,提示PPARγ缺陷将下调巨噬细胞吞噬相关分子的表达。由此证明,PPARγ通过调控巨噬细胞的吞噬相关分子表达影响其吞噬功能。最后,我们用PPARγ特异激动剂罗格列酮(Rosiglitazone,RSG)治疗WT和PPARγ-KO小鼠,发现RSG加快了WT小鼠伤口愈合,并减少了WT伤口TNF-α表达和凋亡细胞数目。然而,RSG对PPARγ-KO小鼠并无治疗效应。说明RSG是通过激动巨噬细胞PPARγ发挥治疗作用,而并不是通过其他非特异性作用。因此,PPARγ对于维持巨噬细胞吞噬凋亡细胞功能促进皮肤伤口愈合至关重要,巨噬细胞PPARγ也将成为今后治疗慢性难愈伤口的重要治疗靶点。
[Abstract]:The skin is the largest organ in the body, to resist the cold stimulation, to prevent pathogen invasion and other important human protection. Because the skin is often exposed to the external environment, easy [1]. skin injury, skin to maintain homeostasis of the body start to repair and restore its structural integrity and normal function of [2]. skin wound healing is a by immune cells. Corneous epithelial cells, fibroblasts, vascular endothelial cells, extracellular matrix, cytokines and growth factors involved in complex multicellular multi medium, physiological process in an orderly manner. Can be divided into tissue inflammation, tissue formation and tissue remodeling stage. Because of the complexity of skin wound healing, healing process susceptible to interference from their own conditions and external environment and obstacles, left chronic refractory wound. To understand the regulation mechanism of normal skin wound healing for clinical treatment Provide a theoretical basis for the treatment of chronic refractory wounds. Macrophages are the regulation of skin wound healing key cell [1,6,7]. macrophages to secrete proinflammatory factor promoting wound inflammation and secretion of protease and active oxygen free radical resistance to pathogens [8-10], secretion and phagocytosis of apoptotic cells to promote wound inflammation and anti-inflammatory factors, secretion of cytokines, growth factors and chemokines the role of epithelial cells in keratinocytes, fibroblasts and vascular endothelial cells to promote wound epithelial regeneration, collagen deposition and angiogenesis of [11]. in wound healing, macrophage chemotactic disorders, impaired phagocytosis of apoptotic cells, abnormal polarization will lead to delayed healing of wound [11-14]. therefore maintain wound macrophages normal function is essential for wound healing. At present, the regulation mechanisms of wound macrophage function is not clear. The nuclear transcription factor peroxide Peroxisome proliferator activated receptors (Peroxisome proliferator activated receptor y, PPAR y [15].PPAR y) is an important molecule regulating macrophage functions can promote macrophage phagocytosis of apoptotic cells and [15,16] macrophages transformed from a proinflammatory state for anti-inflammatory, and promote inflammation subsided [17,18]. PPAR gamma in skin wound healing of macrophages may have regulation effect. Our research is clear and the mechanism of macrophage PPAR gamma in skin wound healing. In this study, we first examined the wild type (Wild, type, WT) expression in mouse wound healing of wound tissue during wound macrophages and PPAR gamma, found after skin injury and tissue macrophages by PPAR expression of macrophage and PPAR gamma indicating that the organization has a moderating effect on wound healing. Then we use Lox P-Cre The system constructs a specific knockdown of macrophage PPAR gamma (PPAR gamma -KO) mice, and the genome contains Lox P loci but not containing Cre enzyme (macrophages PPAR gamma knockout, PPAR gamma -WT) were used as controls. We found that the PPAR gamma -KO mice was significantly delayed wound healing, wound granulation tissue formation, collagen deposition and angiogenesis. We further explore PPAR gamma -KO mice delayed wound healing. Because of inflammatory cells in the normal wound infiltration on wound healing of critical [19-25], we measured the number of neutrophils and macrophages in PPAR gamma -WT and PPAR gamma -KO mice wound, found no significant differences, suggesting that macrophages PPAR gamma knockout does not affect inflammatory cell infiltration. In addition to wound infiltration of inflammatory cells, inflammatory factors also play an important role in wound healing. The wound reported TNF- alpha overexpression in severe loss of Hai Yuhe [26-31]. we found that the expression of PPAR gamma -KO wound TNF- alpha increased significantly, and anti mouse TNF- alpha antagonist in PPAR gamma -KO (Anti-mouse injection wound TNF- alpha, a TNF- alpha) after wound healing was accelerated, granulation tissue formation, collagen deposition and angiogenesis were also increased. Because the macrophage is the main source of cytokines. Tissue, we detected the expression of PPAR gamma -KO wound macrophages TNF- alpha found its expression increased significantly. These results indicate that PPAR gamma -KO wound TNF- alpha overexpression is a key factor in the healing of disorder, and the secretion of PPAR gamma -KO macrophage cells over TNF- alpha promotes wound TNF- expression too much. We further study of the intrinsic mechanism of macrophage PPAR gamma defect of excessive secretion of TNF- induced by alpha. In vitro experiment we found that PPAR -WT and PPAR gamma gamma -KO in resting state were low expression of TNF- in lipopolysaccharide (Li. popolysaccharide,LPS)鍒烘縺涓，

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