CN116570614A - 靶向M2巨噬细胞外囊泡(M2-EVs)lncRNA的组合物及其应用 - Google Patents
靶向M2巨噬细胞外囊泡(M2-EVs)lncRNA的组合物及其应用 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及一种M2巨噬细胞外囊泡(M2‑EVs)lncRNA DACT1‑AS调控ILC2s活化在过敏性哮喘中的应用。靶向M2巨噬细胞外囊泡lncRNA DACT1‑AS的试剂为有效量的减少M2巨噬细胞外囊泡中lncRNA DACT1‑AS表达的试剂,通常为核酸靶向试剂,进一步的,为siRNA或反义寡核苷酸。本发明有效的验证M2‑EVs通过lncRNA DACT1‑AS参与了ILC2s活化的调控,为ILC2s的功能研究提供理论数据,为过敏性哮喘的发病机制和治疗靶点提供理论基础和临床依据。
Description
技术领域
本发明属于生物医药领域,涉及一种靶向M2巨噬细胞外囊泡(M2-EVs)lncRNA的组合物及其通过调控ILC2s活化在过敏性哮喘中的应用。
背景技术
过敏性哮喘(AA)是一种与呼吸系统症状相关的炎症性气道疾病,如呼吸短促、胸闷和咳嗽。近几十年来,哮喘的发病率显著增加,影响全球约3亿人,预计到2025年将影响4亿人,Th2细胞被认为是哮喘发病的主要原因。然而,哮喘是先天免疫和适应性免疫相互作用的结果。这两者相辅相成,共同促进气道炎症的发生。气道组织中的固有免疫细胞群(如M2巨噬细胞和ILC2s)的相互作用在Th2气道炎症的启动、引导和维持中起着非常重要的作用,并逐渐引起关注。
在过去几年中,人们越来越认识到巨噬细胞与2型固有淋巴细胞(ILC2s)的相互作用在各种疾病中发挥着关键作用。通过对环境信号或2型诱导细胞因子(如IL-33)迅速反应,活化的ILC2s能够产生大量的2型细胞因子IL-5和IL-13,这些细胞因子促进嗜酸性粒细胞增多、气道重塑和粘液分泌过多。就巨噬细胞而言,它们可通过一些可溶性细胞因子、细胞与细胞接触和细胞外囊泡(EVs)发挥其免疫调节作用,EVs中蛋白质和酶用于靶向受体细胞并重新编程细胞行为,使EVs的临床潜力显著。最近,M2巨噬细胞衍生的细胞外囊泡(M2-EVs)被鉴定为与肿瘤细胞的侵袭和转移相关,成为炎症和治疗相关的细胞间通讯中的关键信使。然而,目前尚不清楚失调的M2巨噬细胞介导的免疫应答是否通过EVs途径调节ILC2s活化在过敏性气道炎症中发挥致病作用。因此,我们假设ILC2s在过敏性哮喘气道炎症中的激活可由长非编码RNA(lncRNA)调节,因为,lncRNA是EVs最重要的成分之一。
lncRNA是长链非编码RNA,通常被定义为长度超过200个核苷酸的RNA分子,在哺乳动物组织中广泛表达。越来越多的证据表明,lncRNA在一系列生理和病理条件下表现出动态表达模式,并参与调节蛋白质表达与DNA甲基化修饰等。当然,lncRNA在哮喘发病机制中的参与引起了相当大的关注,因为转录组分析显示lncRNA在患有哮喘患者和动物模型外周血中异常表达。因此,M2-EVs中lncRNA可能是改善动物哮喘症状的新治疗靶点。本研究利用微阵列芯片分析发现M2-EVs中同源的lncRNA DACT1-AS(ENSMUST00000132822)高表达,同时在过敏性哮喘小鼠肺组织中也呈高表达。然而,其是否调控ILC2s活化促进过敏性哮喘气道炎症仍不清楚。
发明内容
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
在本发明中,我们发现过敏性哮喘肺组织中存在M2-EVs,M2-EVs可在体内体外促进ILC2s活化。进一步研究发现lncRNA DACT1-AS在M2-EVs中表达增高,敲低M2-EVs中lncRNA DACT1-AS表达后可在体内体外抑制ILC2s活化,同时减少了炎性细胞对过敏性哮喘小鼠气道的浸润程度。因此,本发明有效的验证M2-EVs通过lncRNA DACT1-AS参与了ILC2s活化的调控,为ILC2s的功能研究提供理论数据,为过敏性哮喘的发病机制和治疗靶点提供理论基础和临床依据。
参照图1-11,M2巨噬细胞外囊泡lncRNA DACT1-AS调控ILC2s活化在过敏性哮喘中的应用,其涉及序列号如表1所示。
附图说明
图1:M2巨噬细胞在哮喘气道微环境中分泌EVs。(A)用荧光显微镜检测哮喘小鼠肺组织中的EVs。(B)哮喘肺组织中分离出EVs的TEM图。(B)来自肺组织的EVs的纳米颗粒追踪分析(NTA)的代表性结果。(C)来自肺组织的EVs的CD63、CD81、HSP70和Calnexin的蛋白质印迹分析。(D)通过RT-qPCR测定哮喘肺组织EVs的性质(每组n=10个)。***P<0.001。
图2:M0-EVs和M2-EVs的制备和表征。(A)从巨噬细胞培养基中分离的M0-EVs和M2-EVs的TEM。(B)EVs纳米颗粒追踪分析(NTA)的代表性结果。(C)流式细胞术也证实了EVs的直径。(D)来自M0/M2巨噬细胞和M0/M2 EVs的CD9、CD81、HSP70和Calnexin的蛋白质印迹分析。(E)通过流式细胞术测定EVs中CD63的表达。
图3:M2-EVs在体外显著促进ILC2s的功能。(A)流式细胞仪分析显示,M2-EVs可同时增加IL-5+ILC2s和IL-13+ILC2s的水平,而M0-EVs则不增加(n=4~6)。(B)通过ELISA分析M0-EVs和M2-EVs对刺激的ILC2s中炎性细胞因子水平的影响(n=4~6)。(C)用Ki-67染色分析ILC2s对M0-EVs和M2-EVs的反应。对照组用PBS刺激,数据表示为两个独立实验的平均值±SEM。NS,无显著性,*P<0.05,**P<0.01,***P<0.001。
图4:M2-EVs显著促进小鼠过敏性气道炎症。(A)小鼠过敏性气道炎症发展示意图。(B)H&E染色测定气管周围区域炎性细胞的浸润,PAS染色测定小鼠肺组织上皮杯状细胞的数量(每组n=4~6只小鼠)。(C)对肺组织中ILC2s的流式细胞术分析表明M2-EVs对ILC2s产生的影响比M0-EVs更明显(每组4~6只小鼠)。(D)收集BALF样品,其用于测量所选细胞因子的水平,如ELISA所示(每组4只小鼠)。(E)通过流式细胞术分析肺中IL-5或IL-13阳性ILC2s细胞的百分比(每组n=4~6只小鼠)。数据表示为两个独立实验的平均值±SEM。NS,无显著性,*P<0.05,**P<0.01,***P<0.001。比例尺,H&E染色为100μm,PAS染色为200μm。
图5:ILC2s能够在体外和体内摄取M2-EVs。(A)将小鼠分选的ILC2s与PKH26标记的M2-EVs孵育。最后分析了ILC2s中PKH26的比例。(B)通过荧光显微镜检测ILC2s中的PKH26(红色)。(C)研究肺ILC2s对PKH26标记的M2-EVs的摄取效应的示意图。(D)通过荧光显微镜检测哮喘小鼠肺组织中PKH26标记的M2-EVs。(E)接受PKH26标记的M2-EVs给药的小鼠肺的代表性NIRF图像。(F)流式细胞术分析表明,气管内滴注PKH26标记的M2-EVs后,过敏性气道炎症小鼠肺ILC2s中PKH26标签的M2-EVs的计数显著增加。NS,无显著性,*P<0.05,**P<0.01,***P<0.001。
图6:M0-EVs和M2-EVs之间lncRNA的微阵列分析。(A)热图显示M0-EVs和M2-EVs之间lncRNA的差异表达。(B)通过RT-qPCR确认M0-EVs和M2-EVs之间lncRNA的差异表达。(C)ENSMUST0000132822位于DACT1反义链上。(D)RT-qPCR检测BMDM细胞组分中DACT1-AS水平。U6和GAPDH分别为细胞核和细胞质对照。数据表示为三个独立实验的平均值±SEM。(E)通过RNA-FISH检测M2巨噬细胞中DACT1-AS的表达。比例尺,20μm。
图7:lncRNA DACT1-AS同源性分析。(A)两种lncRNA的GIC分析。(B)ENSMUST00000132822(小鼠)和NONHSAT037154(人类)序列的比对。图8:M2-EVs分泌的DACT1-AS在体外促进ILC2s的活化。(A)RT-qPCR检测M2-EVs中DACT1-AS的表达。(B)M2-EVs和ILC2s共培养48小时后,通过RT-qPCR检测ILC2s中DACT1-AS的表达水平。(C)RNA-FISH检测ILC2s中DACT1-AS的表达。(D)使用专门针对DACT1-AS的抑制剂,在BMDM中敲低DACT1-AS。通过RT-qPCR评估DACT1-AS的表达。使用非靶向siRNA阴性对照(M2-EVs)作为对照。(E)在M2-EVs中DACT1-AS表达被敲除后,通过RT-qPCR检测ILC2s中DACT2-AS的表达。(F)M2-EVs或M2-EVs(DACT1-AS抑制剂)和ILC2s共培养48小时后,用Ki-67染色分析小鼠ILC2s。(G)DACT1-AS敲除对RT-qPCR检测的ILC2s相关基因表达的影响。(H-I)在M2-EVs中抑制DACT1-AS表达后,使用流式细胞术检测ILC2s中的Th2细胞因子水平。(J)在抑制M2-EVs中DACT1-AS的表达并与ILC2s共培养后,收集细胞上清液并通过ELISA检测细胞因子的表达水平。数据表示为两个独立实验的平均值±SEM。NS,无显著性,*P<0.05,**P<0.01,***P<0.001。
图9:DACT1-AS在哮喘小鼠肺组织中的表达。
图10:M2-EVs分泌的DACT1-AS促进体内ILC2s的活化。(A-B)小鼠肺组织的H&E染色和PAS染色(每组n=5-7只小鼠)。(B)通过流式细胞术分析用M2-EVs和M2-EVs(DACT1-AS抑制剂)治疗的哮喘小鼠肺组织中ILC2s的表达(每组n=5-7只小鼠)。(C-E)分析肺组织中IL-5、IL-9或IL-13阳性ILC2s细胞的百分比(每组n=5-7只小鼠)。(F)收集BALF样品,其用于测量所选细胞因子的水平,如ELISA所示(每组n=5-7只小鼠)。(G)通过气道将DiR标记的M2-EVs(DACT1-AS抑制剂)注射到哮喘小鼠体内,并定期对2-3只小鼠实施安乐死,取出其肺部进行体外荧光成像。(H)肺组织中M2-EVs(DACT1-AS抑制剂)荧光强度的变化。数据表示为两个独立实验的平均值±SEM。NS,无显著性,
*P<0.05,**P<0.01,***P<0.001。
图11:DACT1-AS抑制剂对BMDMs表型的影响。BMDMs转染DACT1-AS抑制剂48小时,然后用IL-4(20ng/mL)刺激48小时。从两组细胞中提取RNA,并使用Arg1和iNOS特异性引物进行RT-qPCR分析。GAPDH被设定为内源性对照。数据表示SEM±平均值。结果代表至少两个独立实验,n≥3。*P<.05,***P<0.001。
具体实施方式
下述实施例仅对本发明的技术方案进行说明,不会对本发明的保护范围进行限制。不应被认为是对本发明的范围的限制。实施例中所用的实验方法,如无特殊说明,均为常规方法,所用的培养基、试剂、耗材和试剂盒等,如无特殊说明,均为市售购买产品。
小鼠
Bablc雌性小鼠(6-8周,20-22g)购自青龙山实验动物中心(南京,中国),并饲养在无病原体的SPF级动物房中。所有动物实验均根据《实验动物的护理和使用指南》(中华人民共和国卫生部,1998)和皖南医学院实验动物伦理委员会的指南进行。所有实验方案均由皖南医学院动物伦理委员会评估并批准(许可号:20130138)。
统计分析
所有测得的参数均表示为平均值±SEM。统计软件GraphPad Prism(6.0版)用于分析。使用GraphPad Prism软件(6.0版)进行统计分析。进行了t检验以确定两个独立组之间的差异。P值<0.05被认为具有统计学意义。
实施例1 M2巨噬细胞在哮喘气道微环境中分泌EVs
为了诱导过敏性哮喘,在实验的第0、7和14天,将200μg Papain经腹腔注射给Bablc小鼠。在第21-27天,每天通过雾化器将100μg Papain致敏给小鼠,持续15分钟,并在最后一次雾化后24小时安乐处死小鼠。
EVs根据先前描述的修饰方法通过不同的超速离心从肺中分离出来。简而言之,将肺组织在37℃下在带有275U/mL II型胶原酶溶液的振荡水浴中孵育30分钟。然后通过具有100μm孔的尼龙细胞过滤器过滤细胞悬液,然后在4℃下以300×g连续离心20分钟,3000×g连续离心20分钟和10,000×g60分钟以去除细胞,碎片和大囊泡。将上清液通过0.22μm孔过滤器,然后在贝克曼库尔特Optima XPN-100超速离心机(SW 32转子)中以110,000×g在4℃下超速离心70分钟以产生肺组织的EVs。
将M0或M2巨噬细胞的上清液在4℃下以300g差异离心5分钟,3000g离心20分钟,10,000g离心60分钟以除去细胞和碎片,然后使用0.22μm过滤器过滤。在最后一次离心后,使用贝克曼库尔特Optima XPN-100超速离心机(SW 32转子)在4℃下将上清液进一步超速离心2小时。然后用冰冷的PBS洗涤沉淀,并在4℃下以110,000g超速离心70分钟。最后用100μL PBS悬浮沉淀并用于进一步分析。
随后,对提出的肺组织EVs或者M0-/M2-EVs使用BCA蛋白检测试剂盒确定纯化的肺组织外囊泡的蛋白浓度。Western blot检测CD63,CD81,HSP70和Calnexin等标志物在EVs中的表达。使用粒子跟踪分析仪(Particle Metrix,德国)测量尺寸分布。用TEM观察外泌体的大小和形态。
在第26天,通过连续、差速离心和超速离心步骤从小鼠哮喘模型的肺组织中收集并分离EVs。通过TEM直接检查了分离颗粒的形态特征,颗粒显示为直径约为100nm的杯状膜结合囊泡(图1A)。为了详细说明纯化的颗粒,我们进行NTA以测量囊泡的尺寸分布。几乎所有颗粒的直径都在30至150nm之间,平均值为104±11nm(图1B)。此外,与通过免疫印迹分析的组织裂解物相比,囊泡制剂高度富集了外体标记蛋白HSP70、CD63和CD81,但不富集作为内质网标记物的Calnexin(图1C)。由于M2巨噬细胞在哮喘中占主导地位,检测了从肺组织中提取的EVs含量。与正常小鼠相比,CD206在哮喘小鼠肺组织的EV中高度表达(图1D)。因此,性质分析表明,哮喘小鼠肺组织中释放的分离的囊泡包含M2巨噬细胞的特征。
实施例2M2-EVs促进ILC2s体外活化
ILC2s分选和处理
将BALB/c小鼠的小鼠肺组织在37°c下用含有游离酶(50μg/mL)和DNase I(1μg/mL)的8mL RPMI 1640消化约40分钟。通过70μm细胞过滤器过滤细胞悬浮液,并用RPMI 1640洗涤一次。接下来,按照制造商的说明,使用ILC2s分离试剂盒(STEMCELL Technologies,Canada)纯化BALB/c小鼠的ILC2s。对于ILC2s的鉴定,用FcR阻断试剂(Biolegend,USA)阻断,并用lineage、CD45、CD90和ST2流式抗体进行染色,随后通过流式细胞术(BeckmanCytoflex)进行分析。
为了测定不同EVs对ILC2s的免疫调节作用,将ILC2s以每孔2×105个细胞的速度接种在24孔板中,加入500μL DMEM和10%FBS。然后,在进一步实验之前,用200μg/mL不同的EVs刺激ILC2s约48小时。
PKH26标记EVs再悬浮在无血清培养基中,加入37℃培养箱培养24小时后,用PBS洗涤细胞三次以去除过量的未染上PKH26的EVs。然后,用4%多聚甲醛固定细胞10分钟,用DAPI染色细胞核;使用激光扫描共焦显微镜拍摄照片。同时,ILC2s用lineage、CD45、CD90和ST2流式抗体进行染色,再用Beckman Cytoflex流式细胞仪检测ILC2s中PKH26的比例。
为了研究ILC2s对EVs的体内摄取效应,如上所述,建立了具有过敏性气道炎症的小鼠,在第20天给予PKH26标记的EVs,并在给药后24小时处死。采集肺组织并制备厚度为7μm的冷冻切片。肺切片用丙酮和异丙醇固定,在室温下用2%BSA封闭12小时,然后用DAPI染色。最后,在激光扫描共聚焦显微镜上获得M2-EVs的荧光图像。同时通过流式细胞术分析PKH26标记的EVs在肺ILC2s中的比例。
为了进行进一步的组织学分析,随后将各处理组哮喘小鼠的肺组织取出,并将其在4%多聚甲醛中固定24小时。随后,进行苏木精和曙红(H&E)染色和高碘酸席夫(PAS)染色以评估肺部炎症。
ILC2s细胞因子(IL-5、IL-9和IL-13)和ILC2s中Ki67的核内染色使用eBioscience细胞内固定和渗透缓冲液组试剂和真核转录因子缓冲液组,分别用不同的EVs和Monensin刺激48小时后。然后在4℃下用FcR阻断20分钟后收获ILC2s,并与IL-5、IL-9,IL-13或Ki67流式抗体进行染色,随后通过流式细胞术(Beckman Cytoflex)进行分析。
根据说明书使用ELISA试剂盒(杭州MultiSciences(Lianke)Biotech Co.,Ltd.)对小鼠BALF或者细胞培养上清中IL-5,IL-9和IL-13细胞因子水平进行定量。
由于M2巨噬细胞和ILC2s之间的相互作用在许多疾病中都有报道,我们假设AA中的M2-EVs可以调节ILC2s的免疫应答。因此,为了探讨M2-EVs对ILC2s的影响,我们首先在体外收集了M2-EVs。我们使用超速离心从上清液中分离出的M0-和M2-EVs主要由外泌体组成,但可能受到微泡的污染,因此我们将该系统命名为EVs,以避免以下实验中的任何争论混淆。如图1A–B所示,在TEM和NTA的基础上,分离的M0-和M2-EVs均表现出特征性的“圆形”形貌和均匀尺寸,峰值粒径分别为106.6nm和109.7nm。我们的流式细胞术(CytoFLEX)分析结果也证实了EVs的直径分布在100nm左右(图2C)。使用Western blot,我们进一步证实M0-EVs和M2-EVs对CD9、CD81和HSP70的EV特异性标记物均呈阳性,并且这些标记物在EVs中的水平高于其母细胞。此外,M0-EVs和M2-EVs都缺乏可检测的Calnexin,这是一种在细胞中表达但在EVs中表达较少的内质网膜标记物(图2D)。一致地,EVs的流式细胞术分析显示M0-EVs和M2-EVs上CD63的表面表达(图2E)。总之,EVs在大小、形态和特异性标记方面的特征表明,我们的EVs制剂包括典型的外泌体。
为了评估M2-EVs对ILC2s功能的影响,用M0-EVs和M2-EVs处理健康小鼠肺部的小鼠ILC2s 48小时。我们发现,ILC2s中高水平的IL-5和IL-13被M2-EVs显著激活,但M0-EVs没有(图3A)。虽然M2-EVs处理后ILC2s的IL-9表达水平高于M0-EVs处理后,但无显著差异,因此未显示结果。正如预期的那样,ELISA在ILC2s的条件培养基(CM)中检测到Th2细胞因子在M2-EVs刺激下显著升高,特别是IL-5(图3B)。此外,Ki-67染色证实了M2-EVs介导的对ILC2s增殖的促进作用(图3C)。总的来说,我们的研究结果表明,M2-EVs能够在体外促进ILC2s的功能。
实施例3通过气管内滴注给予M2-EVs加重小鼠过敏性气道炎症
我们确定了M2-EVs是否会影响体内肺ILC2s的功能,我们开发了木瓜蛋白酶诱导的哮喘小鼠模型(图4A)。肺组织的病理分析表明,用M2-EVs处理的小鼠气管周围区域的炎症浸润和上皮杯状细胞的数量均显著增加(图4B)。同样,我们观察到,在暴露于M2-EVs时,小鼠可以发展出严重的肺部炎症,其特征是肺ILC2s的总数增加(图4C),2型效应细胞因子(如IL-5,IL-9和IL-13)的蛋白表达升高(图4D-E)。总之,我们的结果证明了M2-EVs对哮喘小鼠ILC2s活化的影响。
实施例4 ILC2s能够在体外和体内摄取M2-EVs
为进一步了解M2-EVs对ILC2s功能的影响机制,成功制备了PKH26标记的M2-EVs,并评价了ILC2s对M2-EVs体外和体内的摄取作用。我们发现,在流式细胞术处理M2-EVs24小时后,小鼠ILC2s显著吸收了M2-EVs(图5A)。此外,通过使用激光共聚焦荧光显微镜证实了分选小鼠ILC2对M2-EVs(红色)的体外摄取作用(图5B)。另一方面,将PKH26标记的M2-EVs通过气道注射到哮喘小鼠体内,共聚焦图像和NIRF成像结果显示M2-EVs分布在肺组织中(图5C-D)。如图4E所示,注射后M2-EVs在ILC2s中的荧光强度显著较高,证实了M2-EVs对ILC2s靶向活化的影响。总的来说,我们证明了M2-EVs能够在体外和体内的ILC2s中摄取,这对于进一步研究是必要的。
实施例5 M0-EVs和M2-EVs之间lncRNA差异特征的微阵列分析
M0和M2-EVs RNA测序工作由中国广州RiboBio公司完成。EVs的总RNA用于文库制备和测序。RiboBio进行了文库制备和测序。简言之,RNA片段约为200bp。随后,根据Ultra提供的说明,对收集的RNA进行第一链和第二链cDNA合成,然后进行接头连接和低循环富集TMIllumina RNA文库制备试剂盒(NEB,美国)。使用Agilent 2200TapeStation和/>2.0(Life Technologies,USA)对纯化的文库产品进行评估,然后使用HiSeq30000进行测序(2×150bp)。在从原始数据中去除含有适配器、ploy-N和低质量的读数后,获得了干净的读数。HISAT2用于用默认参数将干净的读数与小鼠参考基因组mm10对齐。随后使用HTSeq将对齐的短读转换为每个基因模型的读计数。DEseq使用读取计数作为输入来评估差异表达。Benjamini Hochberg多重测试校正方法已启用。根据|log2(倍数变化)的标准选择差异表达的基因|≥1和p值<0.05。
使用TRIzol试剂(Invitrogen)从细胞EVs中提取总RNA,并使用第一链cDNA合成试剂盒(Applied Biosystems)将提取的RNA用于合成cDNA。然后使用表1中所示的引物在Lightcycler 480系统(Roche)上以cDNA为模板进行qPCR。使用通用反向引物和不同的特异性正向引物进一步扩增所得cDNA,PCR程序如下:在95℃下预变性2分钟,然后在94℃下进行40次循环10秒,58℃下15秒,72℃下20秒,随后进行熔化曲线分析。所有反应一式三份。EVs的mRNA表达水平被标准化为cel-miR-39-5p,而EVs其他的表达水平被标准化为GAPDH,并使用2-ΔΔCt方法计算。
RiboBio(中国广州)合成了针对不同位点DACT1-AS(如SEQ ID NO:9所示)的LncRNA Smart Silencer(一种含有三种ASO和三种siRNA的混合物,如表1所示)。将巨噬细胞以1×106细胞/孔的密度接种在六孔板上过夜,然后使用Lipofectamine 3000(Invitrogen,USA)以100nM的最终浓度转染。转染后再用IL-4刺激48h后通过RT-qPCR检测干扰效率,并选择沉默效率超过70%的Smart Silencer进行进一步实验。
表1:
RNA FISH按照制造商说明书进行。简而言之,将培养的细胞用PBS冲洗一次,并用PBS中的4%多聚甲醛固定20分钟,然后用PBS漂洗三次,用0.2%Triton X-100(Sigma)在PBS中渗透15分钟,用PBS洗涤两次,并用预杂交缓冲液在37℃下孵育30分钟。此后,将细胞在37℃下与含有500nM Cy5标记的DACT1-AS探针的杂交缓冲液孵育17小时。用含0.5%吐温-20的4×SSC连续洗涤三次,以及2×SSC、1×SSC和PBS各洗涤一次后,用DAPI染色细胞以观察细胞核,用PBS冲洗三次。最后,共焦显微镜下进行拍摄。
最近,来自巨噬细胞的EVs通过递送lncRNAs逐渐被认为是不同疾病免疫调节的候选者,但在哮喘中的探索较少。因此,为了鉴定导致M2-EVs对过敏性气道炎症中ILC2s活化影响的lncRNA,我们进行了全lncRNA测序以检测M0-EVs和M2-EVs的RNA表达谱。总的来说,我们在M0-EVs和M2-EVs中鉴定了大约29577个lncRNA。M0-EVs和M2-EVs共有762个lncRNA差异表达,其中299个lncRNA在M2-EVs中表达较高,463个lncRNA在M0-EVs中表达较高(图6A)。为了确认RNA测序结果,选择两种在M2-EVs中高表达的lncRNA通过RT-qPCR进一步确认。如图6B所示,两种lncRNA均富集在M2-EVs中。结果与微阵列数据基本一致。接下来,根据基因重要性计算器(GIC,www.cuilab.cn)和基因保守分析的结果,最终选择了ENSMUST00000132822,其同源基因为NONHSAT037154(图7)。选择ENSMUST00000132822的另一个重要原因是,作为反义lncRNA(图6C),由于核苷酸序列互补性,反义lncRNA对其对应的反义基因具有特殊作用。据报道,ENSMUST00000132822的邻近基因DACT1与巨噬细胞有关,并在哮喘患儿的肺组织中上调。因此,我们将ENSMUST00000132822命名为lncRNA DACT1-AS(SEQ ID NO:9)。总体而言,DACT1-AS被选为介导过敏性气道炎症中M2-EVs激活ILC2s的潜在lncRNA。
为了充分了解DACT1-AS,我们进一步鉴定了DACT1-AS转录本的细胞定位,从M2巨噬细胞(BMDMs)中分离了核和胞质RNA,并测量了DACT1-AS转录本在两个亚细胞位置的表达。RT-qPCR数据显示,与细胞质相比,DACT1-AS转录本在细胞核中高表达(图6D)。作为对照,GAPDH mRNA特异性地位于细胞质中,而U6 RNA主要位于细胞核中。RNA荧光原位杂交(RNA FISH)表明DACT1-AS在细胞核中高表达(图6E)。
实施例6M2-EVs中DACT1-AS在体外促进ILC2s活化
为了测定DACT1-AS抑制后M2-EVs对ILC2s的免疫调节作用,将ILC2s以每孔2×105个细胞的速度接种在24孔板中,加入500μL DMEM和10%FBS。然后,在进一步实验之前,用200μg/mL不同的M2-EVs或M2-EVs(DACT1-AS抑制剂)刺激ILC2s约48小时。
将M2-EVs(DACT1-AS抑制剂)在哮喘雾化激发前一天通过气道注射进入小鼠体内,随后继续雾化3天后安乐死,取肺组织进行流式检测或ELISA检测免疫指标。
DiR标记用于M2-EVs(DACT1-AS抑制剂),研究其在肺组织的时长分布。哮喘小鼠通过气道滴注200μg DIR标记的EVs。然后,使用成像系统(AniView600,广州生物光生物科技有限公司)定时观察肺部的荧光强度。
为了进一步确定M2-EVs中的DACT1-AS是否参与激活气道炎症中的ILC2s,我们再次确定了DACT1-AS的表达(图8A)。在M2-EVs和ILC2s共培养48小时后,我们检测到ILC2s中DACT1-AS的表达水平。RT-qPCR结果显示,ILC2s中DACT1-AS的mRNA表达水平升高(图8B),位于细胞核中(图8C)。同时,我们甚至检测到AA肺组织中DACT1-AS的表达。正如预期的那样,与对照组相比,AA中的DACT1-AS表达增加(图9)。因此,基于上述结果,我们可以知道DACT1-AS至少部分通过M2-EVs在AA中发挥了重要作用。
此外,我们确定了DACT1-AS是否促进了体外ILC2s的活化,我们用DACT1-AS抑制剂降低了其在M2巨噬细胞中的表达,并使用小鼠ILC2s检查了它们的效果。当DACT1-AS的表达下调时,不仅DACT1-AS在M2-EVs中的表达降低,而且DACT1-AS在ILC2s受体细胞中的表达也降低(图8D-E)。接下来,我们研究了抑制DACT1-AS对ILC2s增殖的影响。肺ILC2s的门控策略如图2A所示。ILC2s扩增受到显著抑制,这是通过M2-EVs(DACT1-AS抑制剂)治疗后总肺ILC2s数量的Ki67减少来确定的(图8F)。随后,我们检查了一些与激活ILC2s相关的基因,包括ST2,GATA3,干细胞抗原-1(Sca-1)和诱导性T细胞共刺激器(ICOS)。结果表明,在DACT1-AS抑制作用下,除ICOS外,ST2、GATA3和Sca-1均显著降低。在功能上,在M2-EVs(DACT1-AS抑制剂)处理后,活化的ILC2s(IL-5或IL-9阳性)的百分比也降低了(图8H-I),然而,IL-13阳性ILC2s没有显著降低(数据未显示)。与先前的实验一致,ILC2s上清液中IL-5的分泌减少,但IL-9与对照组相比无显著差异。有趣的是,分泌到细胞上清液中的IL-13量减少(图8J)。总的来说,这些结果强烈表明,M2-EVs中的DACT1-AS激活了先天免疫信号通路,以在体外积极调节ILC2s的激活。
实施例7M2-EVs分泌的DACT1-AS促进体内ILC2s活化。
尽管我们观察到DACT-AS敲低导致ILC2s功能降低,但这提供了进一步的证据,证明M2-EVs中的DACT1-AS可能在AA中起至关重要的作用。为了进一步研究DACT1-AS对体内ILC2s活化的影响,我们使用与前面描述的类似建模方法建立了AA模型。小鼠在第20天用M2-EVs或M2-EVs(DACT1-AS抑制剂)治疗,并在气道雾化后3天获得肺组织。结果表明,与M2-EVs治疗的小鼠相比,用M2-EVs(DACT1-AS抑制剂)治疗的小鼠表现出炎症细胞浸润减少和支气管周围上皮杯状细胞增生(图10A)。然后,我们检查了DACT1-AS对肺组织中ILC2s功能的影响。与我们的上述发现类似,我们观察到与M2-EVs相比,M2-EVs(DACT1-AS抑制剂)对肺组织中ILC2s水平的影响有所减轻(图10B)。更重要的是,除IL-9外,M2-EVs(DACT1-AS抑制剂)扰动显著降低了治疗后ILC2s中IL-5和IL-13的高水平(图10C-E)。在BALF中2型细胞因子(如IL-5)的水平上也发现了类似的结果,而IL-9和IL-13降低但没有产生显着差异(图10F)。
先前的研究表明,M1巨噬细胞的外泌体可以抑制ILC2s的活化,因此,我们推测M2-EVs(DACT1-AS抑制剂)对ILC2s活化的抑制可能与M1巨噬细胞有关。正如我们推测的那样,BMDMs中DACT1-AS转染后IL-4刺激48h,RT-qPCR检测BMDMs显示Arg-1降低,伴有iNOS(M1相关基因)升高(图11)。因此,这表明M2-EVs(DACT1-AS抑制剂)的性质可能由于BMDMs表型改变为M1巨噬细胞而发生了变化。此外,我们还观察了M2-EV(DACT1-AS抑制剂)在体内的持续时间。我们在指定时间对哮喘小鼠实施安乐死,以观察肺组织中DiR标记的M2-EV(DACT1-AS抑制剂)的荧光强度。如图10G-H所示,M2-EVs(DACT1-AS抑制剂)的荧光强度在4h达到峰值后开始下降,并持续4d(96h)或更长时间。
总的来说,这些数据表明M2-EVs加重了小鼠的过敏性气道炎症,这部分归因于M2-EVs中DACT1-AS向ILC2s的递送。
总之,我们的研究结果提供了M2-EVs中DACT1-AS促进ILC2s活化的证据。我们的研究不仅确定了lncRNA通过EVs介导从M2巨噬细胞到ILC2s的细胞间通讯以引起气道炎症的关键机制,而且还为哮喘患者开发了潜在的无创诊断方法和治疗策略。
Claims (7)
1.一种靶向M2巨噬细胞外囊泡的反义lncRNA-β连环蛋白拮抗因子同源物1 (lncRNADACT1-AS)的试剂,其特征在于,其为有效量的减少M2巨噬细胞外囊泡中lncRNA DACT1-AS表达的核酸靶向试剂,选自SEQ ID NO:1-6。
2.根据权利要求1所述的试剂,其特征在于,其包括SEQ ID NO:1-6。
3.一种M2巨噬细胞外囊泡,其特征在于,含有权利要求1或2任一项所述试剂。
4.一种调控2型固有淋巴细胞ILC2s表达的非疾病诊断的方法,其特征在于,施用权利要求1-2任一项所述的试剂。
5.权利要求1-2任一项所述试剂在制备靶向M2巨噬细胞外囊泡lncRNA DACT1-AS的试剂中的用途。
6.权利要求1-2任一项所述试剂或权利要求3所述的巨噬细胞外囊泡在制备治疗炎症性气道疾病的药物中的应用。
7.权利要求6所述应用,其特征在于,其为过敏性哮喘。
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