【作者】 刘芳；

【导师】 段金友；

【作者基本信息】 西北农林科技大学 ， 药学硕士（专业学位）， 2023， 硕士

【摘要】 皮肤光老化和人类对美的追求之间的矛盾日益加深,如何有效防治皮肤光老化是目前科研人员面临的巨大挑战。光老化是由于皮肤长期受到日光照射所引起的损害,通常伴有皮肤松弛、深而粗的皱纹沟壑、干燥脱屑、黑色素细胞和黑色素小体数量增加等症状。外用药物、激光、注射填充等作为预防和治疗皮肤光老化的主要临床手段,存在着刺激性强、渗透性差、价格昂贵等问题,因而开发构建一种高效温和的防治策略刻不容缓。现有研究表明,紫外线辐射是加速皮肤产生氧自由基(Reactive Oxygen Species,ROS)的关键因素,这将破坏细胞的抗氧化能力和皮肤的天然抗氧化屏障系统。外泌体不仅具有免疫调节和组织再生作用,而且因其与原始来源细胞相似的膜结构与内在成分,故此也具有适用于防治皮肤光老化的免疫特权。然而,外泌体容易被机体过早清除的缺点极大地限制了其在再生医学的应用。本文选用三维培养的人真皮成纤维细胞来源的外泌体(3D Human Dermal Fibroblast Cells-Exosomes,HDF-Exo3D)做内容物,利用氧化透明质酸(Hyaluronic Acid,HA)、羧甲基壳聚糖(Carboxymethyl Chitosan,CMCTs)和硫缩酮(Thioketal,TK)交联形成的ROS响应水凝胶支架(CMCTs-TK/HA)包埋外泌体来防治皮肤光老化。利用傅里叶红外光谱、流变学分析和核磁共振氢谱的手段分别从结构和机械性能两个方面证明我们成功制备并筛选出制备浓度为氧化透明质酸(3%w/v)和羧甲基壳聚糖(4%w/v)的水凝胶支架。外泌体释放试验发现水凝胶能有效消耗ROS,且ROS浓度与外泌体释放数量成正比。建立促细胞增殖实验表明包封外泌体后的水凝胶体系具有更高的促细胞增殖能力。通过构建简易小鼠光老化模型对组织中羟脯氨酸含量和胶原蛋白分布作出的定性和定量分析发现,与对照组相比,含有外泌体的水凝胶可直接注射于目标部位或其附近,能够保证更为集中的外泌体剂量,提供持续且明显的治疗效果。研究表明,这种基于氧化透明质酸与羧甲基壳聚糖交联形成的氧化响应型水凝胶(CMCTs-TK/HA@exo3D)是一种极具前景的外泌体缓释型原位给药递送系统,可以防止外泌体过早被清除,提高其生物利用度。同时,为有效改善皮肤光损伤和防治皮肤光老化提供了新思路。更多还原

【Abstract】 The contradiction has deepened day by day between skin photoaging and human’s pursuit of beauty.How to effectively prevent skin photoaging is a great challenge for scientific researchers at present.Photoaging is a long-term skin damage caused by sunlight,which is usually accompanied by skin relaxation,deep and thick wrinkles,dry desquamation,and increased number of melanocytes.Topical drugs,laser,injection and filling are the main clinical methods for the prevention and treatment of skin photoaging,which have some problems such as strong irritation,poor permeability and high price.Therefore,it is urgent to develop and construct an efficient and gentle prevention and treatment strategy.Existing studies have shown that UV radiation is a key factor in accelerating the production of oxygen free radicals(ROS)in the skin,which disrupts the antioxidant capacity of cells and the skin’s natural antioxidant barrier system.Exosomes not only have immune regulation and tissue regeneration functions,but also possess immune privileges suitable for preventing and treating skin photoaging due to their membrane structure and intrinsic composition similar to the original source cells.However,the defect that exosomes are easily cleared by the body prematurely limits its application in regenerative medicine.In this study,the exosomes derived from 3D cultured human dermal fibroblasts(HDFExo3D)were selected as contents,and ROS response hydrogels(CMCTs-TK/HA@exo3D)formed by carboxymethyl chitosan(CMCTS)crosslinking with oxidative hyaluronic acid(HA)and thioketal(TK)were used to encase exosomes in order to prevent and treat skin photoaging.By means of fourier transform infrared spectroscopy,rheological analysis and nuclear magnetic resonance hydrogen spectroscopy,we successfully prepared and screened hydrogel scaffolds with concentrations of oxidized HA(3% w/v)and CMCTS(4% w/v),which could be proved from both structural and mechanical properties.Exosome release tests showed that hydrogels could effectively consume ROS,and the ROS concentration was proportional to the number of exosomes released.The establishment of cell proliferation experiments showed that the hydrogel system encapsulated exosomes had a higher ability to promote cell proliferation.Hydroxyproline content and collagen distribution in tissues were qualitatively and quantitatively analyzed by building a simple mouse photoaging model.Compared with the control group,the hydrogel containing exosomes could be directly injected into the target site,which could ensure a more concentrated exosome dose and provide sustained and obvious therapeutic effect.It has been shown that this oxidation-responsive hydrogel(CMCTs-TK/HA)based on the cross-linking of oxidized hyaluronic acid and carboxymethyl chitosan is a promising targeted exosome delivery system,which can prevent the premature removal of exosomes and improve their bioavailability.At the same time,it provides a new idea for effectively improving skin photodamage and preventing skin photoaging.更多还原