【作者】 张冰；

【导师】 徐欣； 张东姣；

【作者基本信息】 山东大学 ， 口腔医学（专业学位）， 2024， 博士

【摘要】 背景与目的2型糖尿病(type 2 diabetes mellitus,T2DM)是临床最常见的糖尿病类型,是一个在世界范围内发病率较高且对健康极具挑战性的慢性代谢性疾病。随着全球患病率的不断增加,与其相关的并发症例如伤口的愈合受损等也逐渐引起人们的重视。伤口延迟愈合是T2DM主要的并发症之一且难以治疗。口腔是一个多菌环境,因此T2DM患者进行拔牙、种植及其伴随的附加手术等都存在创口愈合延迟的风险,这进一步增加了口腔感染的可能性和严重并发症的易感性。研究T2DM的创口愈合被认为是迄今为止最重要的问题之一。近些年来,T2DM导致创口愈合延迟的机制研究一直是科学研究的热点和难点,寻找能够促进糖尿病患者创口愈合的方法一直是临床亟待解决的问题。因此探究其具体的发病机制并研究更有效的治疗方法提高临床糖尿病患者创口愈合的速度和质量具有重要意义。内质网(Endoplasmic reticulum,ER)作为一种重要的细胞器,在生理上负责钙稳态、蛋白质折叠和脂质生物合成。病理刺激如氧化应激(Oxidative stress,OS)、缺血、钙稳态的破坏等都有助于ER中错误折叠蛋白的积累,从而引起内质网应激(Endoplasmic reticulum stress,ERS)。内质网应激是一个多种病理过程都存在的共同特征,例如神经的变性、炎症、中风和代谢性疾病等。糖脂代谢紊乱会干扰ER的功能并增加ERS。研究发现糖尿病会导致角膜内皮细胞中ERS标志物高于正常组,持续ERS的激活导致角膜内皮损伤,抑制ERS可以改善T2DM角膜内皮功能。自噬是一种细胞内溶酶体相关的降解过程,自噬的调节受各种细胞应激条件的影响,例如OS、ERS、营养和生长因子缺乏以及感染等。ERS的适应性反应是未折叠蛋白反应(Unfolded protein response,UPR)的激活,UPR启动维持内环境稳态。但长时间的应激,UPR不能减轻诱发性应激时,则会引起自噬关键分子的上调和不可逆的细胞损伤和死亡。PERK信号通路作为UPR三个分支之一在ERS中调节保护性生物功能(包括代谢物稳态、细胞氧化还原稳态和线粒体功能)中起重要作用。PERK可以抑制蛋白质翻译,减少蛋白质合成,从而减少ER蛋白质折叠负荷。PERK在ERS时最容易被激活,一旦激活,PERK可以磷酸化真核起始因子 2 的α-亚基(eukaryotic initiation factor 2α,eIF2α),减少其 mRNA 的翻译,同时选择性地增强CHOP和ATF4。ATF4的过度激活会导致CHOP基因的上调,同时增强自噬基因(如ATG5)的表达。因此,探究T2DM是否作为慢性代谢性疾病影响血管内皮细胞的ERS和自噬,最终导致细胞功能障碍的潜在机制十分重要。利拉鲁肽(Liraglutide,Lira)是作为一种长效的胰高血糖素样肽-1(glucagon like peptide-1,GLP-1)受体激动剂的类似物,目前临床上用于治疗T2DM。Lira可用于糖尿病患者的降糖,被认为具有心血管保护作用。除了降糖作用外,Lira还能诱导体重减轻,并具有良好的抗炎、抗氧化和促进血管生成的作用。有研究证明Lira可以通过抑制OS和ERS对糖尿病视网膜神经性变的保护作用。并且有研究报道Lira可以促进人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)的血管生成并且能够保护HUVECs不受棕榈酸盐的损伤。因此推测Lira可能通过调控ERS和OS作为加速糖尿病创面愈合潜在的治疗药物。然而Lira水溶性差、半衰期短以及其在组织中药物浓度不容易维持限制了其临床应用。因此如何维持其给药浓度也是目前待解决的问题之一。聚乳酸-乙醇酸(PLGA)是一种良好的可以用于临床治疗的生物相容性材料,已被美国食品药品监督管理局(US Food and Drug Administration,FDA)批准。由聚乳酸-羟基乙酸-聚乙二醇(PLGA-PEG)纳米颗粒组成的两亲性嵌段共聚物目前已被学者进行广泛研究,此外,其在疾病诊断和治疗方面被作为一种应用广泛的纳米载体体系。PLGA-PEG纳米颗粒(nanoparticles,NPs)可以将药物分子包裹在其形成的纳米颗粒中,同时保持药物活性并实现药物缓释。脱细胞真皮基质(Acellular dermal matrix,ADM)具有良好生物活性和生物相容性,因其去除了高度免疫原性的细胞成分,保留皮肤组织中免疫原性相对较弱的细胞外基质,可以避免局部免疫排斥和强烈的免疫炎症反应。研究发现ADM可以作为良好的组织工程生物支架材料用于创口的组织再生。因此,本研究推测将ADM、NPs和Lira联合应用可能会成为一种新的临床治疗递送策略。综上,本研究首先构建了体内的T2DM大鼠模型及体外模拟T2DM的高糖高脂环境,探究T2DM对皮肤创口愈合和血管内皮细胞生物学功能的影响;其次外源性的加入Lira探究其缓解糖尿病条件下的ERS,保护血管内皮细胞的功能,改善T2DM大鼠的皮肤创口愈合的作用机制;最后通过ADM和NPs联合应用弥补了 Lira的临床应用缺陷,制备了负载Lira纳米颗粒的ADM复合支架(Lira NPs@ADM)作为一种新型的递送系统,探究其在糖尿病创口愈合中的效果,为临床糖尿病患者的治疗提供新的思路。材料与方法1.T2DM对大鼠创口愈合、血管内皮细胞生物学功能及内质网应激标志物表达水平的影响高糖高脂喂养及低剂量腹腔注射链脲佐菌素(Streptozotocin,STZ)联合构建了T2DM大鼠模型,并分别于正常大鼠及T2DM大鼠背部中线两侧制备全层皮肤缺损。分别于术后不同时间点取材并通过苏木素-伊红染色(hematoxylin and eosin,HE),改良Masson 三色染色法(Masson’ strichrome staining,Masson staining)等评价 T2DM 对大鼠创口愈合的影响。此外,利用免疫组织化学染色来检测糖尿病创口愈合中ERS相关因子的表达情况。体外实验培养人脐静脉内皮细胞系,通过联合应用25 mM葡萄糖和200 μM棕榈酸钠模拟体外T2DM的高糖高脂(High glucose and high-fat,HGHL)环境,并观察HUVECs生物学功能的影响。通过CCK-8评价细胞的增殖能力;通过细胞划痕实验和transwell小室检测细胞的迁移能力;通过基质胶成管实验检测细胞的成管能力;利用实时荧光定量 PCR(Quantitative Real-time polymerase chain reaction,qRT-PCR)和 western blot 检测HUVECs的ERS相关因子的表达。2.利拉鲁肽通过缓解内质网应激改善高糖高脂条件下血管内皮细胞损伤和功能障碍的作用和机制体外培养HUVECs,分别经正常培养基、HGHL培养基和外源性加入Lira的HGHL培养基处理,观察细胞生物学功能的影响。包括采用CCK-8筛选细胞适宜的药物浓度;通过EDU荧光染色进行细胞增殖能力的检测;利用细胞划痕实验和transwell小室对细胞的迁移能力进行检测;采用基质胶成管实验检测不同处理下HUVECs的成管能力;经DCFH-DA染色用于对细胞内活性氧(reactive oxygen species,ROS)进行检测;通过qRT-PCR和western blot技术对ERS和自噬相关基因和蛋白的表达情况进行检测。体外培养HUVECs,分别经HGHL培养基、加入Lira的HGHL培养基,加入10 μM GSK2656157(PERK通路特异性抑制剂)的HGHL培养基以及同时加入Lira和GSK2656157的HGHL培养基处理,采用EDU荧光染色、细胞划痕实验和transwell小室进行细胞增殖和迁移能力的比较;采用基质胶成管实验检测不同处理条件下HUVECs的成管能力;采用荧光染色检测细胞内ROS的变化;通过western blot技术和免疫荧光染色对ERS和自噬相关蛋白的表达情况进行检测。3.T2DM创口愈合治疗药物载体脱细胞真皮基质的制备和评价(1)ADM的制备:将猪来源的皮肤组织经过中性蛋白酶及Triton-100等处理后获得无细胞成分的细胞外基质,再经过梯度酒精脱水和漂洗获取ADM,经灭菌后于等渗溶液中4℃保存。(2)ADM的性能评价:通过扫描电子显微镜(scanning electron microscope,SEM)观察ADM的三维空间结构;通过H&E和Masson staining观察ADM支架的组成成分;通过罗丹明-鬼笔环肽荧光染色、活/死细胞染色和CCK-8观察人牙龈成纤维细胞(human gingival fibroblasts,HGFs)在ADM支架上的黏附情况并评价了 ADM的生物相容性。体内实验通过将ADM置于比格犬的犬牙局部粘膜下,通过CS3600数字口内扫描仪获取口内软组织情况,利用Geomagic Qualify Probe软件分析不同材料对牙龈软组织增厚的效果。并于术后不同时间点取材局部牙龈软组织,通过H&E和Masson staining以观察软组织血管和胶原纤维的生成情况。4.负载利拉鲁肽纳米颗粒的脱细胞真皮基质对T2DM大鼠创口愈合的作用(1)Lira NPs制备和评价:采用乳化溶剂蒸发法制备包载Lira的Lira NPs。通过透射电子显微镜(transmission electron microscope,TEM)、动态光散射(dynamic light scattering,DLS)技术、傅里叶变换红外光谱(fourier transform infrared spectrum,FTIR)、和高效液相色谱法(high performance liquid chromatography,HPLC)对 Lira NPs 进行评价。(2)Lira NPs@ADM的制备和评价:将ADM浸泡于上所制备的Lira NPs溶液中,置于摇床上24 h后通过物理吸附得到Lira NPs@ADM复合支架。通过SEM对复合支架的形貌进行分析并检测Lira NPs是否成功加载至ADM上。构建T2DM大鼠模型,并于背部中线两侧制备全层皮肤缺损。实验分别设置空白对照,ADM,Black NPs@ADM和Lira NPs@ADM组,于术后不同时间点拍照并取材组织。通过H&E和Masson staining观察组织再生情况及血管和胶原纤维形成情况;利用免疫组织化学染色观察新生组织中血管生成相关蛋白CD31和α-SMA的表达及ERS和自噬相关蛋白GRP78和P62的表达。结果1.T2DM损害大鼠创口愈合和人脐静脉内皮细胞生物学功能并导致内质网应激标志物GRP78和CHOP表达水平升高高糖高脂喂养及低剂量腹腔注射STZ联合成功构建了 T2DM大鼠模型,三次随机血糖大于16.7 mmol/L,并出现糖尿病典型的“多食、多汗、多尿、体重减轻”的症状。背部皮肤缺损术后5天和14天获取样本组织。数码相机拍摄伤口外观显示与正常组大鼠相比,T2DM组的创口愈合能力明显较差、愈合速度明显降低。H&E和Masson staining结果显示,T2DM组的创口上皮化速度慢且胶原纤维沉积不如正常组广泛。免疫组织化学染色结果显示,T2DM组的ERS相关蛋白GRP78和CHOP的表达明显升高。体外结果显示,在HGHL培养条件下,HUVECs的增殖、迁移和成管能力明显降低。qRT-PCR和western blot结果从基因和蛋白水平上证实了 HGHL条件下ERS相关因子的表达上升。以上结果均提示,T2DM影响创口的愈合和损害血管内皮细胞的生物学功能,同时T2DM能够干扰ER的功能增加ERS的发生。2.利拉鲁肽通过缓解内质网应激改善高糖高脂条件下血管内皮细胞损伤和功能障碍的作用和机制Lira能明显改善HGHL条件下HUVECs细胞的增殖、迁移和成管能力。此外,HGHL条件下HUVECs的ROS明显升高,利拉鲁肽预处理后明显降低ROS的产生,缓解氧化应激的发生。qRT-PCR和western blot结果显示,HGHL条件下GRP78、CHOP以及p-PERK的表达升高和自噬相关的ATG5、LC3II/LC3I升高、P62的表达降低,Lira的加入能够逆转ERS和自噬相关因子的表达。以上结果提示,Lira能够缓解HGHL导致的ERS和自噬的发生,改善HUVECs的功能。PERK通路特异性抑制剂GSK2656157的回复实验结果表明,在HGHL培养基条件下单独加入Lira或GSK2656157和同时加入Lira和GSK2656157都可以改善血管内皮细胞功能,减少ROS的产生,不同处理组对血管内皮细胞功能的影响没有明显差异。此外,不同处理组都可以显著降低HGHL条件下血管内皮细胞的ERS和自噬,HGHL+Lira+GSK2656157组与HGHL+GSK2656157组之间没有统计学差异。以上结果提示Lira可能是通过PERK通路调节ERS和自噬的发生改善血管内皮细胞的功能。3.T2DM创口愈合治疗药物载体脱细胞真皮基质的制备和评价乳白色的ADM膜在SEM下可见胶原纤维的有序排列,H&E和Masson staining显示ADM中无细胞成分,主要为胶原纤维;分离培养的HGFs在ADM上仍有良好的增殖和黏附能力且活/死细胞染色结果显示ADM有良好的生物相容性。体内动物实验显示ADM有良好的软组织增量的效果,同时术后不同时间点的组织染色结果显示ADM在软组织愈合的早期血管数量和密度增加,胶原纤维沉积广泛。以上结果提示本课题组制备的ADM是具有良好生物相容性和稳定性,且具有良好软组织增量效果的生物支架材料。4.负载利拉鲁肽纳米颗粒的脱细胞真皮基质对T2DM大鼠创口愈合的作用成功合成Lira NPs,通过TEM观察Lira NPs呈均匀的球形;通过DLS技术检测Lira NPs的粒径和Zeta电位;用PBS和含有10%血清的细胞培养基模拟生理条件检测合成的Lira NPs具有良好的稳定性;FTIR检测Lira被成功包载在纳米颗粒内;HPLC评价合成的Lira NPs实现了 Lira的缓释。以上结果提示合成的Lira NPs具有良好的稳定性并可以实现良好的药物缓释。最后通过SEM证实了 Lira NPs被成功加载至ADM上形成了新的Lira NPs@ADM复合支架。体内T2DM大鼠皮肤创口愈合实验中,H&E和Masson staining显示Lira NPs@ADM能够明显提高新生组织的血管生成及胶原纤维的沉积,进而促进T2DM大鼠软组织的愈合。免疫组织化学染色结果显示Lira NPs@ADM组新生组织中血管生成相关蛋白CD31和α-SMA的表达增加,GRP78的染色结果显示Lira NPs@ADM组的表达最低,P62的表达最强。以上结果提示Lira NPs@ADM支架具有良好的生物安全性并可能通过调节ERS和自噬促进糖尿病创口的愈合。结论1.T2DM大鼠的创口愈合能力较差,并且HGHL条件下HUVECs的增殖、迁移和成管等能力明显降低。此外,通过体内和体外模拟糖尿病条件均可导致ERS相关因子的表达升高。2.在HGHL培养条件下外源性的加入Lira能够减轻HUVECs的ERS,缓解自噬,改善血管内皮细胞的功能。且Lira对HGHL条件下血管内皮细胞功能的影响可能通过调控ERS相关通路PERK来实现。3.制备的ADM具有良好的生物相容性和生物活性,是可选择的组织工程生物支架材料。4.Lira NPs@ADM能够缓解ERS和自噬,促进血管的生成和胶原纤维的沉积从而明显改善T2DM大鼠创口愈合。更多还原

【Abstract】 Background and objectivesType 2 diabetes(T2DM)is the most common type of diabetes in clinic.It is a chronic metabolic disease with a high incidence rate worldwide and a great challenge to health.With the continuous increase of global disease incidence,related complications such as impaired wound healing have gradually attracted people’s attention.Delayed wound healing is the main complication of T2DM and is difficult to be treated.The oral cavity is a multi bacterial environment,so T2DM patients are at risk of delayed wound healing during tooth extraction,implantation,and accompanying additional surgeries,which further increases the likelihood of oral infections and susceptibility to serious complications.The study of wound healing in T2DM is considered to be one of the most important problems so far.In recent years,the research on the mechanism of delayed wound healing caused by T2DM has been a hot and difficult issue in scientific research.Finding a method that can promote wound healing in diabetes patients has always been an urgent clinical problem.Therefore,it is of great significance to explore its specific pathogenesis and study more effective treatment methods to improve the speed and quality of wound healing in clinical diabetes patients.The endoplasmic reticulum(ER)is an important organelle responsible for calcium homeostasis,protein folding,and lipid biosynthesis in physiology.Pathological stimuli such as oxidative stress(OS),ischemia,and disruption of calcium homeostasis all contribute to the accumulation of misfolded proteins in ER,leading to endoplasmic reticulum stress(ERS).ERS is a common feature of many pathological processes,including neurodegeneration,stroke,metabolic diseases,and inflammation.Dysregulation of glucose and lipid metabolism can interfere with the function of ER and increase ERS.Studies have found that diabetes can cause the ERS markers in corneal endothelial cells to be higher than those in normal groups,and continuous activation of ERS can lead to corneal endothelial damage.Inhibition of ERS can improve T2DM corneal endothelial function.Autophagy is an intracellular lysosome related degradation process,and its regulation is influenced by various cellular stress conditions,such as OS,ERS,nutrient and growth factor deficiencies,and infections.The adaptive response of ERS is the activation of unfolded protein response(UPR),which initiates to maintain homeostasis in the internal environment.However,prolonged stress and UPR cannot alleviate induced stress,which can lead to upregulation of autophagy key molecules and irreversible cell damage and death.As one of the three branches of UPR,PERK signaling pathway for regulating protective biologic functions including metabolite homeostasis,cellular redox homeostasis and mitochondrial function in ERS.PERK can inhibit protein translation,reduce protein synthesis,and thus reduce ER protein folding load.PERK is most easily activated during ERS.Once activated,PERK phosphorylates the eukaryotic initiation factor 2α(eIF2α),reducing its mRNA translation while selectively enhancing CHOP and ATF4.The excessive activation of ATF4 can lead to upregulation of CHOP gene and enhance the expression of autophagy genes(such as ATG5).Therefore,it is important to explore the potential mechanisms by which T2DM,as a chronic metabolic disease,affects cellular autophagy and apoptosis through ERS,ultimately leading to cellular dysfunction.Liraglutide(Lira)is a long-acting glucagon like peptide-1(GLP-1)receptor agonist analog currently used in clinical practice for the treatment of T2DM.Lira can be used to reduce blood sugar in patients with diabetes and is considered to have cardiovascular protection.In addition to its hypoglycemic effect,Lira can also induce weight loss and has good anti-inflammatory,antioxidant,and angiogenic properties.Some studies have proved that Lira can protect retinal nerve deformation in diabetes by inhibiting OS and ERS.And there are research reports that Lira can promote angiogenesis in human umbilical vein endothelial cells(HUVECs)and protect them from damage caused by palmitate.Therefore,we speculate that Lira may be a potential therapeutic drug to accelerate wound healing of diabetes by regulating ERS and OS.However,the poor water solubility,short half-life,and difficulty in maintaining drug concentration in tissues of Lira limit its clinical application.Therefore,how to maintain its drug concentration is also one of the current unresolved issues.Polylactic acid glycolic acid(PLGA)is a material with good biocompatibility and has been approved by the US Food and Drug Administration(FDA)for clinical treatment.The amphiphilic block copolymer composed of polylactic acid hydroxyacetic acid polyethylene glycol(PLGA-PEG)nanoparticles has been widely studied and used as a nanocarrier system for disease diagnosis and treatment.PLGA-PEG nanoparticles(NPs)can encapsulate drug molecules in the formed nanoparticles while maintaining drug activity and achieving drug sustained release.Acellular dermal matrix(ADM)has good biological activity and biocompatibility.Due to its removal of highly immunogenic cellular components and retention of relatively weak immunogenic extracellular matrix in skin tissue,it can avoid local immune rejection and strong immune inflammatory reactions.Research has found that ADM can serve as a good tissue engineering scaffold material for tissue regeneration in wounds.Therefore,we speculate that the combined application of ADM,nanoparticles,and Lira may become a new clinical treatment delivery strategy.In summary,this study first established an T2DM rat model in vivo and simulated the environment of high glucose and high-fat in vitro to investigate the effects of T2DM on skin wound healing and vascular endothelial cell biological function;Secondly,the exogenous addition of Lira was used to explore its mechanism of relieving ERS under the condition of diabetes,protecting the function of vascular endothelial cells,and improving the skin wound healing of rats in T2DM;Finally,the clinical application defects of Lira were compensated for by the combined application of ADM and nanoparticles.An ADM composite scaffold loaded with Lira nanoparticles(Lira NPs@ADM)was prepared as a new delivery system,which is explored its effect in the healing of diabetes wounds.Lira NPs@ADM may provide new ideas for clinical treatment of diabetes patients.Materials and methods1.The effects of type 2 diabetes mellitus on wound healing,biological function of vascular endothelial cells and expression of endoplasmic reticulum stress markers in ratsT2DM rat models were constructed by combining high sugar and high-fat feeding with low-dose intraperitoneal injection of streptozotocin(STZ),and full-thickness skin defects were prepared on both sides of the midline of the back in normal rats and rats with T2DM.Samples were collected at different postoperative time points and evaluated for the effect of T2DM on wound healing in rats with hematoxylin and eosin(HE)staining and modified Masson’s trichrome staining(Masson’s staining).In addition,the expression of ERS related factors was detected with immunohistochemical staining in diabetes wound healing.Experiments were conducted to cultivate human umbilical vein endothelial cell lines in vitro.The high glucose and high-fat(HGHL)environment of T2DM was simulated by combining 25 mM glucose and 200 μM sodium palmitate,and the biological functions of HUVECs were observed.Evaluate the proliferation ability of cells through CCK-8:Detecting the migration ability of cells through cell scratch test and transwell chambers;Detecting the tube forming ability of cells through matrix gel tube forming experiments;The expression of ERS-related factors in HUVECs was dected with real time polymerase chain reaction(qRT-PCR)and western blot.2.The role and mechanism of liraglutide in improving vascular endothelial cell injury and dysfunction under HGHL conditions by alleviating endoplasmic reticulum stressHUVECs were cultured in vitro and treated with normal medium,HGHL medium,and HGHL medium supplemented with exogenous Lira to observe the effects on cellular biological functions.Including using CCK-8 to screen for suitable drug concentrations for cells;Detect the proliferation ability of cells through EDU fluorescence staining;Experiments of cell scratch and transwell chambers were used to detect cell migration ability;Matrix gel tube formation experiment was used to observe the tube forming ability of HUVECs under different treatments;DCFH-DA staining was used to detect changes in reactive oxygen species(ROS)within cells;The expression of ERS and autophagy related genes and proteins was detected through qRT-PCR and western blot.HUVECs were cultured in vitro and treated with HGHL medium,HGHL medium containing Lira,HGHL medium containing 10 μM GSK2656157(specific inhibitors of PERK pathway),and HGHL medium containing both Lira and GSK2656157.The changes in cell proliferation and migration ability were compared using EDU fluorescence staining,cell scratch experiments,and transwell chambers;The tube forming ability of HUVECs was tested with matrix gel tube forming experiments under different treatments;Using fluorescence staining to detect changes in intracellular ROS;The expression of ERS and autophagy related proteins was detected with western blot technique and immunofluorescence staining.3.Preparation and evaluation of acellular dermal matrix as a drug carrier for type 2 diabetes mellitus on wound healingtreatment(1)Preparation of ADM:Skin tissue from pigs was treated with neutral protease and Triton-100 to obtain extracellular matrix without cellular components.Then,ADM was obtained by gradient alcohol dehydration and rinsing,sterilized,and stored at 4℃ in isotonic solution.(2)Performance evaluation of ADM:Observing the three-dimensional spatial structure of ADM through scanning electron microscopy(SEM);Observing the composition of ADM scaffolds through H&E and Masson staining;The adhesion of human gingival fibroblasts(HGFs)on ADM scaffolds was observed and the biocompatibility of ADM was evaluated using rhodamine phalloidin fluorescence staining,live/dead staining,and CCK-8.In vivo experiments were conducted by placing ADM under the local mucosa of the canine teeth of beagle dogs,obtaining the soft tissue situation inside the mouth through the CS3600 digital intraoral scanner,and analyzing the effect of different materials on gingival soft tissue thickening using Geomagic Qualify Probe software.Local gingival soft tissues were collected at different postoperative time points,and H&E and Masson staining were used to observe the generation of blood vessels and collagen fibers in the soft tissues.4.The effect of acellular dermal matrix loaded with liraglutide nanoparticles on wound healing in type 2 diabetes mellitus rats(1)Preparation and evaluation of Lira NPs:Lira NPs loaded were prepared by emulsified solvent evaporatio technology.Lira NPs were evaluated using transmission electron microscopy(TEM),dynamic light scattering(DLS),Fourier transform infrared spectroscopy(FTIR),and high performance liquid chromatography(HPLC).(2)Preparation and evaluation of Lira NPs@ADM:ADM was immersed in the Lira NPs solution and placed on a shaker for 24 hours to obtain Lira NPs@ADM composite scaffold through physical adsorption.Analyze the morphology of the composite scaffold through SEM and detect whether Lira NPs have been successfully loaded onto ADM.T2DM rat models were onstructed and full-thickness skin defects were prepared on both sides of the midline of the back.Experiments were conducted with blank controls,ADM,and Black NPs@ADM and Lira NPs@ADM.Take photos and tissue samples at different time points after surgery.Observe tissue regeneration and the formation of blood vessels and collagen fibers through H&E and Masson staining;Using immunohistochemical staining to observe the expression of angiogenesis related proteins CD31 and α-SMA,as well as the expression of ERS and autophagy related proteins GRP78 and P62 in newborn tissues.Results1.T2DM impaired wound healing in rats and biological function of HUVECs,led to increased expression levels of ERS markers GRP78 and CHOPThe T2DM rat models were successfully established by feeding with high sugar and high-fat diet and intraperitoneal injection of low-dose STZ.The blood glucose was more than 16.7 mmol/L for three times at random,and the typical symptoms of diabetes,such as"overeating,sweating,urination and weight loss" were observed.Sample tissues were obtained at 5 and 14 days after surgery from back skin defects.The appearance of the wound captured by a digital camera showed that compared with the normal group of rats,the T2DM group had significantly poorer wound healing ability and slower healing speed.The H&E and Masson staining results showed that the epithelialization rate of wounds in the T2DM group was slower and collagen fiber deposition was not as extensive as in the normal group.The results of immunohistochemical staining showed that the expression of ERS proteins GRP78 and CHOP was significantly increased in the T2DM group.The results in vitro showed that the proliferation,migration,and tube forming ability of HUVECs were significantly reduced under HGHL culture conditions.The qRT-PCR and western blot results confirmed the upregulation of ERS related factors expression under HGHL conditions at the gene and protein levels.The above results all suggested that T2DM affect wound healing and damage the biological function of vascular endothelial cells.At the same time,T2DM could interfere with the function of ER and increase the occurrence of ERS.2.The role and mechanism of liraglutide in improving endothelial cell injury and dysfunction under HGHL conditions by alleviating endoplasmic reticulum stressLira could significantly improve the proliferation,migration,and tube forming ability of HUVECs cells under HGHL conditions.In addition,ROS levels in HUVECs were significantly increased under HGHL conditions,and pretreatment with lira significantly reduced ROS production,alleviating the occurrence of OS.The results of qRT PCR and western blot suggested that the expression of GRP78,CHOP,and p-PERK increased,while the expression of autophagy related ATG5,LC3II/LC3I increased and P62 decreased under HGHL conditions.The addition of Lira was able to reverse the expression of ERS and autophagy related factors.The above results suggest that Lira can alleviate the occurrence of ERS and autophagy caused by HGHL,and improve the function of HUVECs.The response experiment results of PERK pathway specific inhibitor GSK2656157 showed that the addition of lira or GSK2656157 alone,as well as the simultaneous addition of lira and GSK2656157 under HGHL medium conditions,could improve endothelial cell function and reduce ROS production.There was no significant difference in the effect of different treatment groups on endothelial cell function.In addition,different treatment groups could significantly reduce ERS and autophagy in endothelial cells under HGHL conditions.There was no statistical difference between the HGHL+Lira+GSK2656157 group and the HGHL+GSK2656157 group.The above results suggested that Lira may improve endothelial cell function by regulating ERS and autophagy through the PERK pathway.3.Preparation and evaluation of acellular dermal matrix as a drug carrier for type 2 diabetes mellitus on wound healingtreatmentThe milky white ADM membrane showed ordered arrangement of collagen fibers under SEM.H&E and Masson staining showed no cellular components in ADM,mainly collagen fibers;The isolated and cultured HGFs still had good proliferation and adhesion ability on ADM,and the results of live/dead staining showed that ADM had good biocompatibility.The animal experiments had shown that ADM had a good effect on soft tissue augmentation in vivo,and tissue staining results at different time points after surgery showed that ADM increased the number and density of blood vessels in the early stage of soft tissue healing,with extensive deposition of collagen fibers.The above results indicated that the ADM prepared by our research group was a biocompatible and stable biological scaffold material with good soft tissue increment effect.4.The effect of acellular dermal matrix loaded with liraglutide nanoparticles on wound healing in type 2 diabetes mellitus ratsLira NPs were synthesized and successfully and were observed as uniform spherical shapes with TEM;Detecting the particle size and Zeta potential of Lira NPs using DLS technology;The synthesized Lira NPs showed good stability when were tested under simulated physiological conditions in PBS and cell culture medium containing 10%serum;FTIR was used to detect the successful encapsulation of Lira in NPs;The synthesized Lira NPs were evaluated using HPLC to achieve sustained release of Lira.The above results indicated that the synthesized Lira NPs have good stability and can achieve good drug sustained release.Finally,SEM confirmed that Lira NPs were successfully loaded onto ADM to form a new Lira NPs@ADM composite scaffold.H&E and Masson staining showed Lira NPs@ADM could significantly enhance angiogenesis and collagen fiber deposition in newly formed tissues,thereby promoting soft tissue healing in T2DM rats through skin wound healing experiment of T2DM rat in vivo.The immunohistochemical staining results showed that the expression of angiogenesis related proteins CD31 and α-SMA in Lira NPs@ADM group increased in newly formed tissues,and the staining results of GRP78 and P62 showed the GRP78 expression in Lira NPs@ADM group is the lowest,while the expression of P62 is the strongest.The above results indicated that Lira NPs@ADM composite scaffold had good biological safety and may promote the healing of diabetes wounds by regulating ERS and autophagy.Conclusions1.The wound healing ability of T2DM rats was poor,and the proliferation,migration,and tube forming ability of HUVECs were significantly reduced under HGHL conditions.In addition,simulating diabetes conditions in vivo and in vitro could lead to increased expression of ERS related factors.2.Exogenous addition of Lira under HGHL culture conditions could alleviate ERS in HUVECs,alleviate autophagy and improve endothelial cell function.The effect of Lira on endothelial cell function under HGHL conditions was mainly achieved by regulating the ERS related pathway PERK.3.The prepared ADM had good biocompatibility and biological activity,and was an optional tissue engineering biological scaffold material.4.Lira NPs@ADM could alleviate ERS and autophagy,and promote angiogenesis and collagen fiber deposition,thereby significantly improve wound healing in T2DM rats.更多还原