【作者】 王丽；

【导师】 刘镇宁；

【作者基本信息】 吉林大学 ， 仿生科学与工程， 2024， 博士

【摘要】 由烧伤、撕裂以及顽固性皮肤溃疡等造成的皮肤损伤是一个世界性的公共健康问题。皮肤伤口愈合是一个动态有序的生物学过程,可分为四个不同的阶段:止血期、炎症期、增殖期和重塑期。在这一过程中,炎症反应和免疫微环境被认为是影响伤口愈合的重要因素。临床上多采用医用敷料来帮助伤口愈合。在临床治疗过程中的伤口炎症反应主要包括三个方面:损伤造成的应激性炎症反应,由细菌感染导致的炎症反应,以及医用敷料引发的免疫排异炎症反应。临床上可以采用抗生素降低细菌感染导致的炎症反应,但针对应激性炎症反应和医用敷料引发的免疫排异炎症反应仍缺乏有效控制手段。亚精胺(SPD)是一种最初在精液中发现的天然活性多胺,可以调控精子作为“异物”所引发的免疫反应,从而促进哺乳动物受精过程和胚胎着床。本论文受此启发,拟利用SPD的免疫调控功能抑制医用敷料作为“异物”引发的免疫排异炎症反应。同时,基于SPD对巨噬细胞极化的调控作用,本论文拟通过在医用敷料中应用SPD缓解伤口的整体炎症反应,改善伤口的免疫微环境,从而加速伤口愈合。聚碳酸亚丙酯(PPC)是一种由二氧化碳和环氧丙烷合成的脂肪族聚碳酸酯,近年来被视为一种有效利用温室气体二氧化碳的环境友好型材料。但由于PPC的疏水性,与细胞、组织的低黏附性,单纯的PPC难以作为伤口治疗的医用敷料;并且作为合成高分子,PPC也将引发免疫排异炎症反应。受细胞外基质(ECM)组成成分启发,本论文拟通过壳聚糖(CTS)和明胶(GEL)对PPC进行改性,从而提高其生物相容性。但从虾壳中获得的CTS和从猪皮中获得的GEL对于人和本论文中的实验动物大鼠而言仍属于外源性物质,故仍将引发免疫排异炎症反应。因此,本论文拟在针对PPC的生物改性过程中应用SPD,开发基于PPC的仿生抗炎医用敷料,拓展环境友好型材料在医疗领域里的应用。首先,以伤口湿性愈合理论为指导,在CTS与GEL的混合溶液中加入甘油(Gly),采用流延法制备了一系列CTS-GEL-Gly(CGG)膜。结合溶胀测试和拉伸测试结果,确定了最优Gly添加量为2.5%(v/v)。CGG2.5膜具有良好的机械性能,克服了CTS-GEL膜延展性差、易碎的缺陷。其次,基于对苯二甲醛(TA)醛基与SPD氨基之间的希夫碱反应构建了SPD浓度分别为0.0125、0.0250、0.0375、0.0500、0.0625、0.0750、0.0875和0.1000M的SPD交联剂。其中,TA与SPD的摩尔浓度之比为1.2:1.0。该配比保证了交联剂上的剩余醛基能再次与CGG膜中的氨基发生交联反应,由此成功制备了化学交联负载SPD的CGG膜,即SCGG膜。然后,采用等离子体处理和旋涂法制备了以PPC作为中间层,SCGG膜作为上、下层的SCGG-PPC-SCGG(SPS)复合膜。尤为重要的是,本论文选择与SPD化学结构相似的二乙烯三胺(DETA)作为SPD的对照,按照相同制备流程制备了负载DETA的DCGG-PPC-DCGG(DPD)复合膜。物理和化学表征结果表明,SPS和DPD复合膜都具有明显的“三明治”结构、良好的溶胀性、适宜的水蒸气透过性、优异的力学性能和热稳定性。体外生物学测试结果表明,当交联剂浓度范围介于0.0375-0.0750 M时,SPS和DPD复合膜具有良好的细胞相容性、促划痕愈合性和广谱抗菌性。然而,炎症标志物检测结果表明,SPS复合膜能使促炎标志物白介素6(IL-6)、肿瘤坏死因子α(TNF-α)和一氧化氮(NO)的浓度分别降低66.19%、52.37%和71.86%,使抗炎标志物白介素10(IL-10)的浓度提高48.21%。并且SPS复合膜对炎症标志物的抑制或促进作用依赖于SPD浓度。相比而言,虽然DPD复合膜也表现出一定的效果,但其效果不具有DETA浓度依赖性,而呈波动趋势。此外,分化抗原簇分子206/一氧化氮合酶(CD206/i NOS)免疫荧光双染结果表明,SPS复合膜能促进Mφ2巨噬细胞标志物CD206表达,抑制Mφ1巨噬细胞标志物i NOS表达。而DPD复合膜无法达到相同效果。综合来看,负载SPD的SPS复合膜具有体外抗炎免疫调控功能,能通过诱导Mφ1巨噬细胞极化为Mφ2巨噬细胞发挥其抗炎作用。本论文构建了SD大鼠全层皮肤缺损模型,以探究SPS复合膜的伤口愈合能力以及体内抗炎免疫调控性能。与未治疗组(Blank)、PPC组(PPC膜)和DPD组(DPD-6膜)相比,SPS复合膜治疗组(SPS-6膜)的伤口愈合最快且效果最好,其再生皮肤没有过度的Ⅰ型胶原蛋白沉积,更接近正常皮肤组织。分化抗原簇分子11b(CD11b)、分化抗原簇分子163/分化抗原簇分子68(CD163/CD68)和i NOS/CD68免疫荧光染色结果表明,SPS复合膜不仅可以抑制伤口炎症和免疫细胞浸润,而且还能调控巨噬细胞表型,促进促炎性Mφ1巨噬细胞向促修复性Mφ2巨噬细胞极化。该结果也被蛋白免疫印迹(Western blot)证实,即SPS复合膜能显著抑制伤口愈合过程中促炎细胞因子,如核因子κB(NF-κB)、单核细胞趋化蛋白1(MCP-1)、IL-6、白介素2(IL-2)和TNF-α的表达;促进抗炎细胞因子,如转化生长因子(TGF-β)和IL-10的分泌;并刺激表皮和真皮再生相关生长因子,如表皮生长因子(EGF)、成纤维细胞生长因子(b-FGF)和血管内皮生长因子(VEGF)的表达。最后,为了更深层次探讨SPS复合膜促Mφ2巨噬细胞极化机制和促伤口愈合机制,本论文对术后第7天采集的Blank组、SPS组和DPD组的伤口组织进行了蛋白质组学分析。通过三组对比,即SPS vs.DPD,SPS vs.Blank和DPD vs.Blank,分析了SPS复合膜影响巨噬细胞极化的机制。结果表明负载SPD的SPS复合膜通过抑制乙酰辅酶A羧化酶1(Acaca)的表达,一方面抑制AMPK/NF-κB信号通路,从而抑制促炎细胞因子表达;另一方面,通过促进三羧酸循环(TCA)和脂肪酸氧化(FAO)代谢,加速伤口部位早中期Mφ1巨噬细胞向Mφ2巨噬细胞极化。负载SPD的SPS复合膜可通过上述两方面作用抑制伤口炎症反应和调控伤口免疫微环境,从而加速伤口愈合,并使再生皮肤更贴近正常皮肤。更多还原

【Abstract】 Skin wound has become a serious healthcare issue worldwide due to the increasing incidence of burns,tears,and chronic disease-caused skin ulcers.Wound healing is a dynamic biological process that can be divided into four different stages: hemostasis,inflammation,proliferation,and remodeling.Inflammatory response and immune microenvironment play important roles in wound healing.Wound dressings are commonly used to facilitate wound healing in clinics.The clinical inflammatory response of wounds is mainly contributed by three reasons: injury-induced stress,bacterial infection,and foreign body rejection(FBR)against medical dressings.Antibiotics can be used to reduce inflammation caused by bacterial infection.However,there remains a grand need for effective measures to control stress-induced inflammation and FBR-inflammation associated with medical dressings.Spermidine(SPD)is a biogenic polyamine originally identified in semen and later found to modulate immune responses to protect sperms,which are regarded as “foreign bodies” by the female immune system,to facilitate fertilization and embryo implantation.Inspired by the immunomodulatory function of SPD,this dissertation proposes to inhibit the inflammatory response triggered by wound dressing with SPD.Meanwhile,based on the regulatory effect of SPD on macrophage polarization,a wound dressing containing SPD will be developed to alleviate overall inflammatory response,improve immune microenvironment,and thus accelerate wound healing.Polypropylene carbonate(PPC),an aliphatic polycarbonate synthesized from carbon dioxide and propylene oxide,has been regarded as an environment-friendly material.However,due to the hydrophobicity,low cell and tissue adhesion of PPC,pure PPC cannot be used as a wound dressing directly,and as a synthetic polymer,PPC will also trigger FBR.Inspired by the composition of extracellular matrix(ECM),this dissertation plans to modify PPC with chitosan(CTS)and gelatin(GEL)to improve its biocompatibility.However,CTS obtained from shrimp shells and GEL obtained from pig skin are still exogenous to rats used in this work and humans,so they will still trigger FBR.Therefore,this dissertation intends to develop bio-inspired antiinflammatory dressing by introducing SPD into the biomodification of PPC,and expand the application of environment-friendly materials in medical fields.First,after the addition of glycerol(Gly)to the mixture of CTS and GEL a series of CTS-GEL-Gly(CGG)films have been prepared by casting,which is guided by the theory of moist wound healing.According to the results of swelling test and tensile test,the optimal Gly content is determined as 2.5%(v/v).The CGG2.5 film shows good mechanical property,which overcomes the defects of poor ductility and fragility of CTS-GEL film.Second,a crosslinker based on the Schiff base reaction between the amino groups of SPD and the aldehyde groups of terephthalaldehyde(TA)has been prepared with SPD concentrations of 0.0125,0.0250,0.0375,0.0500,0.0625,0.0750,0.0875 and0.1000 M,which is denoted as SPD crosslinker.The molar ratio of TA to SPD is 1.2:1.0,which ensures that the remaining aldehyde groups of the crosslinker can undergo another Schiff base reaction with amino groups in the CGG film.Thus,the SPD-loaded CGG film,i.e.,SCGG film,has been successfully prepared.Next,SCGG-PPC-SCGG(SPS)composite film has been fabricated with PPC as the mid-layer and SCGG films as the two outside layers by plasma treatment and spincoating.More importantly,diethylenetriamine(DETA),a sham molecule resembling SPD in chemical structure,has been chosen as a control for SPD,and DETA-loaded DCGG-PPC-DCGG(DPD)composite film has been prepared in parallel.The physicochemical characterizations show that both SPS and DPD composite films have obvious "sandwich" structures,good swelling properties,suitable water vapor permeability,excellent mechanical strength and thermal stability.Moreover,in vitro biological tests indicate that SPS and DPD composite films possess good cytocompatibility,scratch healing function,and broad-spectrum antibacterial properties within the crosslinker concentration of 0.0375-0.0750 M.However,the in vitro results of inflammatory assays exhibit that SPS composite film can reduce the levels of proinflammatory markers such as interleukin 6(IL-6),tumor necrosis factor alpha α(TNF-α),and nitric oxide(NO)by about 66.19%,52.37%,and 71.86%,respectively,and increase the concentration of anti-inflammatory marker interleukin 10(IL-10)by about48.21%.Such effects of SPS composite film also depend on the concentration of SPD.In contrast,DPD film shows a little favorable effect that fluctuates independent of the concentration of DETA.Besides,the double immunofluorescent staining against cluster of differentiation 206/inducible nitric oxide synthase(CD206/i NOS)displays that SPS composite film can promote the expression of Mφ2 macrophage marker CD206,and inhibit the expression of Mφ1 macrophage marker i NOS,while DPD film cannot achieve the same outcome.Taken together,SPS film containing SPD has demonstrated in vitro anti-inflammatory and immunomodulatory functions,while promoting the polarization of Mφ1 macrophage to Mφ2 macrophage.Then,full-thickness skin wound model has been established in SD rats and the wounds were treated with pure PPC films(PPC group),SPS films(SPS-6 group)and DPD films(DPD-6 group)to examine the wound healing and immunomodulatory properties in vivo.Compared to the untreated group(Blank),PPC group and DPD-6group,the SPS-6 group shows faster healing process and better healing outcome with no excessive deposition of type Ⅰ collagen,which is closer to the normal skin.The results of immunofluorescent staining against cluster of differentiation 11b(CD11b),cluster of differentiation 163/cluster of differentiation 68(CD163/CD68),and i NOS/CD68 display that SPS film can not only inhibit inflammation and immune cell infiltration in wound bed,but also accelerate the macrophage polarization from proinflammatory Mφ1 to repairing Mφ2.The results are also corroborated by Western blots,which exhibit that SPS film can significantly down-regulate the expression of pro-inflammatory cytokines including nuclear factor kappa-B(NF-κB),monocyte chemoattractant protein-1(MCP-1),IL-6,interleukin 2(IL-2),and TNF-α,and enhance the secretion of anti-inflammatory factors,such as transforming growth factor β(TGF-β)and IL-10.Meanwhile,SPS film can also up-regulate the expression of grow factors involved in epidermal and dermal regeneration,i.e.epidermal growth factor(EGF),fibroblast growth factor(b-FGF)and vascular endothelial growth factor(VEGF).Finally,in order to explore the mechanism for SPS film to promote Mφ2macrophage polarization and wound healing,proteomic analysis has been conducted on the tissues of Blank,SPS and DPD groups,which were collected on postsurgical Day 7.The mechanism for the effect of SPS film on macrophage polarization has been investigated by mutual comparison among the three groups,i.e.SPS vs.DPD,SPS vs.Blank,and DPD vs.Blank.The results suggest that,on one hand,SPD-loaded SPS film can down-regulate the expression of acetyl Co A carboxylase 1(Acaca)and thus inhibit the AMPK/NF-κB signaling pathway to suppress the expression of pro-inflammatory cytokines;and on the other hand,SPS film can enhance tricarboxylic acid cycle(TCA)and fatty acid oxidation(FAO),thereby accelerating the transition from Mφ1macrophage-dominated phase to Mφ2 macrophage-dominated phase during wound healing.Hence,SPD-loaded SPS film can reduce inflammatory response and modulate immune microenvironment through the above two aspects to promote wound healing and yield regenerated skin closer to normal skin.更多还原