【作者】 何川；

【导师】 龙洁；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2022， 博士

【摘要】 干细胞来源多样且具有自我更新及分化潜能,因此被广泛应用于组织工程及再生医学的研究并已实现多项干细胞治疗的临床应用转化。传统理论认为干细胞主要通过分化为某些特定类型的细胞来增加或替代局部受损的组织从而实现机体解剖结构和生理功能的恢复,然而,研究发现植入体内的干细胞可能没有足够长的寿命来实现自我增殖并向特定的组织细胞分化,且其应用还面临着诸如免疫排斥反应、成瘤和栓塞的潜在可能。随着对干细胞促进组织修复再生机制研究的深入,其扮演的复杂角色逐渐被认知——除了向靶组织类型的细胞分化外,干细胞还能通过分泌具有抗凋亡、促进血管生成和免疫调节作用的生物活性因子发挥作用。已有越来越多的研究致力于分离干细胞的分泌产物用于组织再生修复和疾病治疗,即无细胞治疗(Cell Free Therapy),其核心是以干细胞作为治疗分子的来源,而不是作为治疗剂本身,实现组织再生修复或疾病治疗的目标,这一理念是对干细胞治疗的补充和对组织修复再生策略的丰富。无细胞治疗的基础是分泌组(Secretome)——一类特定状态下细胞分泌到胞外空间的分子及生物因子的集合,主要分为可溶性因子(Soluble factors)和细胞外囊泡(Extracellular Vesicles,EV)。实际研究中,可溶性因子以条件培养基(Conditioned Medium,CM)为载体,与EV一起作为两种主要的无细胞治疗研究对象得到广泛关注。研究证实即使是相同来源的条件培养基与EV在作用机制和应用方面也可能存在差异。本课题组前期研究发现脂肪组织浸提液(Adipose Tissue Extract,ATE),即脂肪组织的条件培养基和从ATE中纯化的小细胞外囊泡(Small Extracellular Vesicles Derived From Adipose Tissue,sEV-AT)均具有促进血管和脂肪组织再生的作用,但尚未对二者进行系统的比较。因此,本课题旨在对同属脂肪组织来源的sEV-AT和ATE在促进组织修复再生的机制及效率等方面的差异进行系统比较以明确二者各自的特点,这对于临床应用无细胞治疗实现组织再生具有重要的指导意义。此外,在进行脂肪组织相关研究的过程中,研究者发现真皮脂肪层与毛发有紧密的联系,其厚度随毛囊的自发周期循环而发生显著变化,并且已有多项动物实验及临床试验证实局部应用脂肪及其衍生物具有促进毛发生长的作用,然而脂肪的直接应用存在仅限自体应用、保存困难、感染及栓塞风险等局限,因此,本研究在明确两种脂肪组织来源的衍生物sEV-AT和ATE具有促进脂肪组织再生作用的基础上,继续观察二者是否具有促进毛发再生的作用,以期利用无细胞治疗的优势为脱发治疗的临床应用提供更多选择。基于课题组对sEV-AT和ATE的前期研究基础,本课题在第一部分研究内容中首先对sEV-AT和ATE的蛋白质得率、颗粒浓度、粒径分布及蛋白质表达谱进行比较;在第二部分研究内容中在等蛋白浓度的前提下比较了sEV-AT和ATE对包括脂肪组织来源的间充质干细胞(Rat Adipose Derived Stem Cell,r ASC)、人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell,HUVEC)和小鼠巨噬细胞系RAW264.7三类脂肪组织再生相关功能细胞的调控作用;在第三部分研究内容中首先比较了sEV-AT和ATE对真皮乳头细胞(Mouse Dermal Papilla Cell,m DPC)增殖、迁移和生长因子分泌的影响,其次观察了sEV-AT和ATE在体外对离体小鼠触须毛囊生长的影响,最后研究了sEV-AT和ATE在体内对小鼠背部毛发生长的作用。所取得的主要研究成果如下:1.本研究系统比较了sEV-AT和ATE的蛋白质得率、颗粒浓度、粒径分布及蛋白质表达谱,结果显示ATE的蛋白质得率显著高于sEV-AT;sEV-AT富集大量粒径集中的小细胞外囊泡,而ATE仅含有极少量粒径分散的囊泡但富含可溶性蛋白;蛋白质谱分析提示sEV-AT和ATE均有促进细胞增殖、迁移和组织再生的潜力,且sEV-AT可能比ATE有更显著的生物学功能。2.本研究比较了sEV-AT和ATE在促进脂肪组织再生相关功能细胞的增殖、迁移能力、r ASC的成脂能力、HUVEC的成血管能力及RAW264.7的趋化能力方面的差异。研究表明sEV-AT与ATE均能够促进体外脂肪组织再生相关功能细胞的各项生物学功能,且在同等蛋白浓度的前提下sEV-AT的作用更高效,而ATE较sEV-AT的提取有更便捷的过程、更低的成本及更高的蛋白质得率等特点,同样具有临床应用的价值。因此sEV-AT与ATE二者鲜明的特点和各自的优势可为后期将脂肪组织来源的衍生物应用于临床提供重要的选择参考。3.本研究首次观察了sEV-AT和ATE对m DPC增殖、迁移能力和生长因子表达能力的影响。结果显示sEV-AT与ATE在体外显著促进m DPC中FGF7的表达和分泌但不能促进m DPC的增殖、迁移和细胞周期进程;sEV-AT或ATE与离体小鼠触须毛囊共培养均能促进毛囊毛干的生长和黑色素沉积,但在同等蛋白浓度的前提下sEV-AT促进离体小鼠触须毛囊毛干生长和黑色素沉淀的作用更显著;同时sEV-AT还能够显著促进离体毛囊真皮乳头区FGF7的表达。皮下注射sEV-AT和ATE促进了小鼠背部脱毛区域的毛发生长,加快了毛囊从静止期到生长期的转换;在等蛋白质浓度前提下,sEV-AT促进小鼠毛发生长的效果更显著。综上,本研究对sEV-AT和ATE进行了系统的比较,明确了二者的基本特征,比较了二者在体外应用于脂肪组织再生相关功能细胞的效率。此外,本研究首次将sEV-AT和ATE用于促进毛发再生的研究,证实了这两种脂肪组织无细胞衍生物具有促进小鼠毛发生长的作用。尽管sEV-AT在多个方面的表现优于ATE,但ATE较sEV-AT具有更便捷的提取过程、更低的提取成本及更高的蛋白质得率,二者特征鲜明且各具优势,可为后期在无细胞治疗的临床应用中对脂肪组织衍生物的选择提供重要参考。更多还原

【Abstract】 Stem cells have been studied extensively in tissue engineering and regenerative medicine because of their diverse sources,self-renewal and differentiation potential,and many results have been clinically applied and transformed into stem cell therapies.In conventional theory,it is widely accepted that stem cells realize the reconstruction of anatomical structure and physiological function by differentiating into some specific cell types to increase or replace the local damaged tissue.However,the tracing experiments of implanted stem cells in vivo indicated that mesenchymal stem cells might have too short life span to accomplish self-proliferation and differentiation into specific tissue cells.What is more,its application is also faced with the problems of potential immune rejection,tumor formation and embolism.As the understanding of the mechanism of stem cells promoting tissue repair regeneration moved forward,the complex role of stem cells play in tissue repair has been recognized.In addition to differentiating into specific tissue cell types,stem cells could also play a therapeutic role by secreting bioactive factors with anti-apoptotic,proangiogenic and immune regulating effects.Nowadays,more and more researchers are trying to isolate stem cells secretory products for tissue repair and treatment of diseases,named as Cell Free Therapy.Cell Free Therapy focuses on the stem cells as the source of therapeutic molecules,rather than drug itself,to realize the goal of tissue regenerative repair or illness treatment.The idea is to complement stem cell therapy and to enrich strategies of tissue repair and regeneration.The fundamental element of cell free therapy is secretome,a collection of the molecules and bioactive factors secreted by cells into extracellular space under specific conditions,which could be categorized into soluble factors and extracellular vesicles(EV).In practical research,soluble factors are carried by conditioned medium(CM)and have received prevalent attention as main cell free therapy research objects together with EV.It has been proved by research that even when CM and EV from the same source could show differences in their function mechanisms and applications.Our preliminary study found that both adipose tissue extract(ATE),conditioned medium of adipose tissue and small extracellular vesicles derived from adipose tissue(sEV-AT)purified from ATE,can promote the regeneration of blood vessels and adipose tissue.However,no systematic comparisons have been made.Therefore,this study aims to systematically compare the differences between sEV-AT and ATE in the mechanism and efficiency of promoting tissue repair and regeneration,so as to clarify their respective characteristics,which have important guiding significance for the clinical application of cell-free therapy to achieve tissue regeneration.In addition,in the adipose tissue related studies,researchers found that the thickness of dermal adipose layer is closely related to hair,which changes significantly with the spontaneous cycle circulation of hair.Moreover,a number of animal experiments and clinical trials have confirmed that the local application of adipose and its derivatives can promote hair growth.However,the direct application of adipose is limited to autologous application,difficulty in preservation,risk of infection and embolism,etc.Therefore,this study continued to observe whether the two derivatives of adipose tissue,sEV-AT and ATE,could promote hair regeneration on the basis of confirming that they could promote tissue regeneration,in the hope to make use of the advantages of cell-free therapy to provide more options for clinical application of alopecia treatment.Based on our previous research on sEV-AT and ATE,the protein yield,particle concentration,particle size distribution and protein expression patterns of sEV-AT and ATE were compared in the first part of the study.In the second part,the regulation of sEV-AT and ATE on adipose tissue regeneration related cells including adipose tissuederived mesenchymal stem cells,human umbilical vein endothelial cells and mouse macrophage RAW264.7 was compared under the premise of the same protein concentration.In the third part of the study,the effects of sEV-AT and ATE on dermal papilla cell proliferation,migration and growth factor secretion were first compared.Then the effects of sEV-AT and ATE on the growth of hair follicles ex vivo were observed.Finally,the effects of sEV-AT and ATE on hair growth in vivo were studied.The main research results are as follows:1.In this study,the protein yield,particle concentration,particle size distribution and protein expression patterns of sEV-AT and ATE were systematically compared.The protein content of ATE per gram of adipose tissue was significantly higher than that of sEV-AT.sEV-AT was enriched with a large number of small extracellular vesicles with consistent particle size,while ATE possessed more soluble proteins.Protein profiling analysis suggested that both sEV-AT and ATE had the potential to promote cell proliferation,migration and tissue regeneration.2.This study compared the differences between sEV-AT and ATE in promoting the proliferation and migration ability of adipose tissue regeneration-related cells,the adipogenic ability of r ASC,the angiogenic ability of HUVEC and the chemotaxis ability of RAW264.7.Both sEV-AT and ATE could promote various biological functions of cells related to adipose tissue regeneration in vitro,and sEV-AT has a stronger effect under the premise of the same protein concentration.Given that ATE has more convenient extraction process and lower extraction cost of conditioned medium and the high cost and complicated process in preparation,and inconvenient storage of EV,conditioned medium,especially tissue-derived conditioned medium,may be a feasible strategy for clinical application.3.This study was the first to observe the effects of sEV-AT and ATE on proliferation,migration and growth factor expression of m DPC.sEV-AT and ATE significantly promoted the expression and secretion of FGF7 in m DPC in vitro but did not promote the proliferation,migration and cell cycle progression of m DPC.Both sEV-AT and ATE co-culture could promote the growth and melanin deposition of murine vibrissa follicle ex vivo,but sEV-AT played a stronger effect under the premise of the same protein concentration.Meanwhile,sEV-AT can significantly promote the expression of FGF7 in dermal papillary region of murine vibrissa follicle.Subcutaneous injection of sEV-AT and ATE promoted the transformation of hair cycle from telogen to anagen and accelerated hair growth of murine dorsal skin.Also,sEVAT had a more significant promoting effect under the premise of the same protein concentration.In summary,this study compared sEV-AT and ATE systematically,and clarified their basic characteristics and their efficiency of applying to adipose tissue regeneration related cells in vitro.In addition,this study confirmed that these two cellfree derivatives of adipose tissue have the effect of promoting murine hair growth.Although sEV-AT performed better than ATE in many aspects,ATE had its own advantages,such as more convenient extraction process,lower extraction cost and higher protein yield,which provided an important reference for the selection of adipose tissue derivatives in the clinical application of cell-free therapy in the future.更多还原