【作者】 徐志红；

【导师】 李银萍；

【作者基本信息】 武汉大学 ， 病理学与病理生理学， 2022， 博士

【摘要】 背景:急性肺损伤（Acute lung injury,ALI）是临床常见急危重症,严重时导致急性呼吸窘迫综合征,威胁患者的生命。以巨噬细胞、中性粒细胞为主的免疫细胞介导的炎症反应失调及炎症因子风暴是ALI的主要发病机制。目前,临床尚缺乏针对ALI的有效、副作用低的治疗方法。间充质干细胞（Mesenchymal stem cells,MSCs）具有强大的免疫调节和抗炎功能,可抑制肺部炎症,减轻肺部损伤;并且这些功能与MSCs的旁分泌功能密切相关。低氧预处理、基因修饰、3D培养等可提升MSCs旁分泌产物的干预效果。然而,目前缺少一种既能提高MSCs的扩增产量和细胞质量,又能有效提升MSCs及其旁分泌产物免疫治疗效果的培养方式。体内干细胞巢为低氧环境,且体外培养体系中的持续性低氧环境有利于维持干细胞的干性并促进其增殖。但持续性低氧培养对MSCs的免疫调节功能、在ALI中的免疫治疗效果及其机制尚不清楚。目的:探讨持续性低氧培养对脐带间充质干细胞（Umbilical cord mesenchymal stem cells,UC-MSCs）分泌组（条件培养基和外泌体）改善ALI作用的影响及其潜在分子机制。方法:通过组织块贴壁法分别将人脐带组织置于常氧（21%O₂）培养箱和低氧（1%O₂）培养箱中培养,获取的细胞分别为常氧MSCs（Nor-MSCs）和低氧MSCs（Hypo-MSCs）。对比分析Nor-MSCs和Hypo-MSCs的增殖、干性、衰老以及免疫调节相关因子分泌的情况,评估持续性低氧培养对UC-MSCs产量和细胞质量的影响。进一步收集Nor-MSCs和Hypo-MSCs来源的条件培养基（Nor-CM和Hypo-CM）,通过流式细胞术和免疫荧光评估Hypo-CM对小鼠巨噬细胞极化和胞葬功能的影响。构建ALI小鼠模型并分别给予两种条件培养基干预,通过HE染色、流式细胞术、免疫荧光等方法评估Hypo-CM在ALI中的干预效果以及对肺内巨噬细胞极化和胞葬功能的影响。收集鉴定Nor-CM和Hypo-CM中的外泌体（Nor-Exo和Hypo-Exo）,通过流式细胞术、ELISA和免疫荧光评估Hypo-Exo对小鼠巨噬细胞和临床ALI患者来源巨噬细胞的极化及胞葬功能的影响。构建ALI小鼠模型并给予外泌体干预,评估Hypo-Exo在肺损伤中的干预效果以及对肺内巨噬细胞极化和胞葬功能的影响。接下来,通过mi RNA测序分析两种外泌体的显著差异性mi RNA,并筛选出mi R-30c-5p。分析Hypo-Exo中mi R-30c-5p敲低或Nor-Exo中mi R-30c-5p过表达对外泌体调控小鼠巨噬细胞极化的影响。进一步构建ALI模型并给予mi R-30c-5p敲低的Hypo-Exo或mi R-30c-5p过表达的Nor-Exo干预,评估mi R-30c-5p在Hypo-Exo干预肺损伤中的作用及对肺内巨噬细胞极化和胞葬功能的影响。最后,通过RT-q PCR和免疫荧光,探究Hypo-Exo对肺组织中Notch1通路活化的影响,并明确mi R-30c-5p在Hypo-Exo调控肺内巨噬细胞Notch1通路中的作用。结果:与Nor-MSCs相比,Hypo-MSCs的增殖能力和干性均增强,且衰老细胞数量较低。同时,Hypo-MSCs分泌更高水平的免疫调节相关因子。Nor-CM与Hypo-CM均能在体内外促进小鼠抗炎型巨噬细胞极化、增强巨噬细胞的胞葬功能,并改善ALI模型肺损伤、抑制肺部炎症;与Nor-CM相比,Hypo-CM的作用更强。进一步对外泌体的研究发现,Hypo-Exo在促进小鼠或人巨噬细胞向抗炎型巨噬细胞极化、增强巨噬细胞胞葬功能中的效果均优于Nor-Exo,且Hypo-Exo改善ALI模型肺损伤中的效果亦更强。外泌体mi RNA测序结果以及RT-q PCR证实,mi R-30c-5p在Hypo-Exo中的表达水平显著高于Nor-Exo。当Hypo-Exo中mi R-30c-5p的表达降低后,Hypo-Exo促进抗炎型巨噬细胞极化的作用减弱;而Nor-Exo中mi R-30c-5p的过表达可增强Nor-Exo的调控作用。ALI模型的实验结果表明,敲低mi R-30c-5p后,Hypo-Exo抑制肺部炎症、改善小鼠肺部损伤、调控抗炎型巨噬细胞极化和胞葬功能的作用均减弱。同时,Nor-Exo与Hypo-Exo均能抑制肺组织中Notch1信号通路相关分子的表达,且Hypo-Exo的作用更强;敲低mi R-30c-5p能削弱Hypo-Exo对ALI模型肺组织中Notch1通路活化的抑制作用。结论:持续性低氧培养既能提高UC-MSCs体外扩增的产量和质量,还能促进UC-MSCs分泌免疫调节相关因子。UC-MSCs分泌组在体内外均能促进抗炎型巨噬细胞极化、提高巨噬细胞胞葬功能,并有效减轻ALI模型肺部炎症、改善肺损伤;并且,持续性低氧培养体系来源的Hypo-CM和Hypo-Exo的效应较Nor-CM和Nor-Exo更为显著。mi R-30c-5p是介导Hypo-Exo调节巨噬细胞极化和胞葬功能、改善肺损伤的重要功能性成分;且mi R-30c-5p的作用与其负向调控巨噬细胞中Notch1通路活化有关。更多还原

【Abstract】 Background: Acute lung injury(ALI)is a common critical disease,which can lead to acute respiratory distress syndrome(ARDS)in severe cases,and threatens the lives of patients.The main pathogenesis of ALI is the dysregulation of inflammatory responses mediated by immune cell,mainly including macrophages and neutrophils.At present,there are still no effective treatments with low side effects for ALI.Mesenchymal stem cells(MSCs)have strong immunomodulatory and anti-inflammatory functions,and could inhibit lung inflammation as well as reduce lung injury.Moreover,the immunomodulatory functions of MSCs are closely associated with their paracrine products.Some strategies,such as hypoxia preconditioning,gene modification,and 3D culture,can improve the effects of MSCs-derived secretome.However,strategies are scarce,which cannot only enhance the production and quality of MSCs,but also the the immunomodulatory function of MSCs and their secretome.Studies demonstrated that stem cell niche was low in oxygen tension(hypoxia),and culture in hypoxic condition helped to maintain the stemness and promote the proliferation of stem cells.However,effects of consecutive hypoxic culture on immunomodulatory propertity of MSCs,as well as on their therapeutic efficacy in ALI,remain largely unknown.Objective: To investigate the effects of consecutive hypoxia on the therapeutic efficacy of secretome of UC-MSCs in attenuating ALI,as well as the underlying molecular mechanisms.Methods:UC-MSCs were cultured under normoxic culture or hypoxic culture,and they were termed as Nor-MSCs or Hypo-MSCs respectively.The proliferation,stemness,senescence-associated phenotype of UC-MSCs,as well as levels of immunomodulatory factors secreted by UC-MSCs were analyzed.Conditioned medium of UC-MSCs(MSCs-CM)was collected,and termed as Nor-CM and Hypo-CM respectively.Effects of Hypo-CM on polarization and efferocytic capacities of mice macrophages were analyzed using flow cytometry and immunofluorescence.The ALI model was established and administered with MSCs-CM via intratracheal(it).The effects of Hypo-CM on lung inflammation,polarization and efferocytic capacities of macrophages were analyzed by HE staining,flow cytometry,and immunofluorescence.Next,the exosomes from Nor-CM and Hypo-CM were collected(termed as Nor-Exo and Hypo-Exo respectively).Effects of Hypo-Exo on mediating polarization and efferocytic capacities of mice or human macrophages were confirmed using flow cytometry,ELISA and immunofluorescence.Moreover,the Nor-Exo and Hypo-Exo were administered to ALI mice to evaluate the functions of exosomes in alleviating lung inflammation,and modulating polarization and efferocytosis of lung macrophages.Finally,mi RNAs of exosomes were analyzed through mi RNAs sequencing.mi RNA(mi R-30c-5p)was screened out to be potential candidate mediating the effects of UC-MSCs exosomes.The functions of mi R-30c-5p in mediating the polarization of macrophages in vitro or alleviating ALI in vivo were explored through mi R-30c-5p overexpression or knockdown in Nor-Exo or Hypo-Exo.We also investigated changes in the activity of Notch1 pathway in lung macrophages following changes in the expression level of mi R-30c-5p in MSCs-Exo.Results:The Hypo-MSCs exhibited higher proliferation ability,stemness,less senescent cells,and apoptotic cells compared with Nor-MSCs.Moreover,Hypo-MSCs secreted higher levels of immunomodulatoryry mediators than Nor-MSCs.The effects of Hypo-CM in promoting anti-inflammatory polarization and efferocytic capacities in macrophages were more potent than Nor-CM.Similarly,Hypo-Exo was more effective than Nor-Exo in mediating anti-inflammatory polarization of mice or human macrophages as well as alleviating lung injuries.The results of mi RNA-seq and RT-q PCR showed that the level of mi R-30c-5p in Hypo-Exo was significantly higher than Nor-Exo.Knockdown of mi R-30c-5p in Hypo-Exo led to decreased efficacy of Hypo-Exo in alleviating ALI,as demonstrated by lower levels of anti-inflammatory macrophages,aggravated lung injuries and inflammation in ALI models.Moreover,knockdown of mi R-30c-5p led to decreased ability of Hypo-Exo in inhibiting Notch1 signaling.Conclusion:Consecutive hypoxia not only enhanced the quality and output of UC-MSCs in vitro,attenuates cell senescence,but also promoted the secretion of immunomodulatory mediators.Secretome from UC-MSCs modulated polarization and efferocytotic capabilities of anti-inflammatory macrophages,and alleviated lung inflammation.The secretome from Hypo-MSCs(Hypo-CM and Hypo-Exo)exhibited superior advantages over that from Nor-MSCs.Consecutive hypoxic culture promoted expression of mi R-30c-5p in exosomes from UC-MSCs.Hypo-Exo delivered mi R-30c-5p into macrophages to inhibit Notch1 signaling,which contributed to the polarization of anti-inflammatory macrophage,and limited lung inflammation.更多还原