本文選題：皮膚傷口愈合　切入點(diǎn)：巨噬細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：皮膚是人體最大的器官,具有抵御冷熱刺激,阻止病原體入侵等重要人體保護(hù)功能。由于皮膚常常暴露在外界環(huán)境中,極易損傷[1]。皮膚損傷后,機(jī)體啟動(dòng)修復(fù)維持皮膚自身穩(wěn)態(tài),恢復(fù)其完整結(jié)構(gòu)和正常功能[2]。皮膚傷口愈合是一個(gè)由免疫細(xì)胞,角質(zhì)上皮細(xì)胞,成纖維細(xì)胞,血管內(nèi)皮細(xì)胞,細(xì)胞外基質(zhì),細(xì)胞因子以及生長(zhǎng)因子等多細(xì)胞多介質(zhì)共同參與,多階段有序進(jìn)行的復(fù)雜生理過程。可以分為組織炎癥期,組織生成期和組織塑形期。正因?yàn)槠つw傷口愈合的復(fù)雜性,愈合進(jìn)程極易受自身?xiàng)l件和外界環(huán)境的干擾而出現(xiàn)障礙,遺留下慢性難愈傷口。了解正常皮膚傷口愈合的調(diào)控機(jī)制可以為臨床治療慢性難愈傷口提供理論依據(jù)。巨噬細(xì)胞是調(diào)控皮膚傷口愈合的關(guān)鍵細(xì)胞[1,6,7]。巨噬細(xì)胞分泌促炎癥因子促進(jìn)傷口炎癥反應(yīng),分泌蛋白酶和活性氧自由基抵抗病原體[8-10],分泌抗炎癥因子和吞噬凋亡細(xì)胞促進(jìn)傷口炎癥消退,以及分泌細(xì)胞因子,生長(zhǎng)因子和趨化因子等作用于角質(zhì)上皮細(xì)胞,成纖維細(xì)胞和血管內(nèi)皮細(xì)胞促進(jìn)傷口上皮再生,膠原沉積和血管生成[11]。在傷口愈合中,巨噬細(xì)胞趨化障礙,吞噬凋亡細(xì)胞功能受損,極化異常等都將導(dǎo)致傷口遷延不愈[11-14]。因此,維持傷口巨噬細(xì)胞正常功能對(duì)傷口愈合至關(guān)重要。目前,對(duì)傷口巨噬細(xì)胞功能的調(diào)控機(jī)制還不清楚。核轉(zhuǎn)錄因子過氧化物酶體增殖物激活受體(Peroxisome proliferator activated receptorγ,PPARγ)是調(diào)控巨噬細(xì)胞功能的重要分子[15]。PPARγ可以促進(jìn)巨噬細(xì)胞吞噬凋亡細(xì)胞[15,16]并使巨噬細(xì)胞從促炎癥狀態(tài)轉(zhuǎn)化為抗炎癥狀態(tài),進(jìn)而促進(jìn)炎癥消退[17,18]。提示PPARγ在皮膚傷口愈合中對(duì)巨噬細(xì)胞可能具有一定的調(diào)控作用。我們的研究在于明確巨噬細(xì)胞PPARγ在皮膚傷口愈合中的作用及機(jī)制。在本研究中,我們首先檢測(cè)了野生型(Wild type,WT)小鼠傷口愈合過程中傷口組織以及傷口巨噬細(xì)胞PPARγ的表達(dá)變化,發(fā)現(xiàn)皮膚損傷后組織和巨噬細(xì)胞PPARγ表達(dá)上調(diào),提示組織和巨噬細(xì)胞PPARγ對(duì)傷口愈合具有調(diào)節(jié)作用。然后我們采用Lox P-Cre系統(tǒng)構(gòu)建了特異敲除巨噬細(xì)胞PPARγ(PPARγ-KO)小鼠,同時(shí)將基因組含有Lox P位點(diǎn)但不含Cre酶(無巨噬細(xì)胞PPARγ敲除,PPARγ-WT)的小鼠作為對(duì)照。我們研究發(fā)現(xiàn),PPARγ-KO小鼠傷口愈合明顯延遲,傷口肉芽組織生成,膠原沉積和血管生成減少。我們進(jìn)一步探究PPARγ-KO小鼠傷口愈合延遲的原因。因?yàn)檠装Y細(xì)胞在傷口的正常浸潤對(duì)傷口愈合至關(guān)重要[19-25],我們?cè)赑PARγ-WT和PPARγ-KO小鼠傷口中檢測(cè)了中性粒細(xì)胞和巨噬細(xì)胞的數(shù)目,發(fā)現(xiàn)沒有明顯差異,提示巨噬細(xì)胞PPARγ敲除不影響傷口炎癥細(xì)胞浸潤。除炎癥細(xì)胞浸潤外,炎癥因子在傷口愈合中也具有重要作用。文獻(xiàn)報(bào)道傷口TNF-α過度表達(dá)嚴(yán)重?fù)p害愈合過程[26-31]。我們研究發(fā)現(xiàn)PPARγ-KO傷口TNF-α表達(dá)明顯增多,并且在PPARγ-KO傷口注射抗小鼠TNF-α拮抗劑(Anti-mouse TNF-α,a TNF-α)后傷口愈合明顯加快,肉芽組織生成,膠原沉積和血管生成也明顯增加。由于巨噬細(xì)胞是傷口組織細(xì)胞因子的主要來源,我們檢測(cè)了PPARγ-KO傷口巨噬細(xì)胞TNF-α表達(dá)后發(fā)現(xiàn)其表達(dá)明顯增加。這些結(jié)果說明PPARγ-KO傷口TNF-α過多表達(dá)是其愈合障礙的關(guān)鍵因素,并且PPARγ-KO巨噬細(xì)胞分泌的過多TNF-α促使了傷口TNF-α表達(dá)過多。我們進(jìn)一步研究了PPARγ缺陷引起巨噬細(xì)胞分泌過多TNF-α的內(nèi)在機(jī)制。在體外實(shí)驗(yàn)中我們發(fā)現(xiàn),PPARγ-WT和PPARγ-KO在靜息狀態(tài)下都低表達(dá)TNF-α。在脂多糖(Lipopolysaccharide,LPS)刺激下,TNF-α表達(dá)明顯增多但兩者間沒有差異,說明PPARγ對(duì)巨噬細(xì)胞分泌TNF-α沒有直接調(diào)控作用。然而,在LPS刺激下加入凋亡胸腺細(xì)胞(Apoptotic thymocytes,ATs)后PPARγ-WT巨噬細(xì)胞TNF-α表達(dá)明顯減少,而PPARγ-KO巨噬細(xì)胞TNF-α表達(dá)并沒有減少,由于巨噬細(xì)胞吞噬凋亡細(xì)胞后TNF-α表達(dá)會(huì)減少,因此提示PPARγ-KO巨噬細(xì)胞吞噬凋亡細(xì)胞功能障礙。在LPS,ATs和細(xì)胞松弛素B(Actin-filament polymerisation-blocking agent cytochalasin B)共同孵育下,PPARγ-WT和PPARγ-KO巨噬細(xì)胞都持續(xù)高表達(dá)TNF-α,說明TNF-α表達(dá)減少確由吞噬引起,并且巨噬細(xì)胞吞噬二醋酸羧基熒光素(CFSE)標(biāo)記ATs實(shí)驗(yàn)進(jìn)一步證明了PPARγ-KO巨噬細(xì)胞吞噬率下降。在體內(nèi)實(shí)驗(yàn)中,我們發(fā)現(xiàn)PPARγ-KO傷口凋亡細(xì)胞聚集,沒有得到及時(shí)有效的吞噬清除。這些結(jié)果證實(shí)了PPARγ缺陷導(dǎo)致巨噬細(xì)胞吞噬凋亡細(xì)胞功能障礙。巨噬細(xì)胞吞噬功能與其吞噬相關(guān)受體和調(diào)理素(Opsonin)直接相關(guān),我們檢測(cè)了PPARγ-WT和PPARγ-KO巨噬細(xì)胞吞噬受體CD36,Mertk以及調(diào)理素Mfge8,Gas-6,C1qa,C1qb和C1qc的表達(dá),結(jié)果表明PPARγ-KO巨噬細(xì)胞其吞噬相關(guān)分子表達(dá)下降,提示PPARγ缺陷將下調(diào)巨噬細(xì)胞吞噬相關(guān)分子的表達(dá)。由此證明,PPARγ通過調(diào)控巨噬細(xì)胞的吞噬相關(guān)分子表達(dá)影響其吞噬功能。最后,我們用PPARγ特異激動(dòng)劑羅格列酮(Rosiglitazone,RSG)治療WT和PPARγ-KO小鼠,發(fā)現(xiàn)RSG加快了WT小鼠傷口愈合,并減少了WT傷口TNF-α表達(dá)和凋亡細(xì)胞數(shù)目。然而,RSG對(duì)PPARγ-KO小鼠并無治療效應(yīng)。說明RSG是通過激動(dòng)巨噬細(xì)胞PPARγ發(fā)揮治療作用,而并不是通過其他非特異性作用。因此,PPARγ對(duì)于維持巨噬細(xì)胞吞噬凋亡細(xì)胞功能促進(jìn)皮膚傷口愈合至關(guān)重要,巨噬細(xì)胞PPARγ也將成為今后治療慢性難愈傷口的重要治療靶點(diǎn)。
[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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