CN113005093A - Cd73修饰的人脐带血来源的外泌体及其应用 - Google Patents
Cd73修饰的人脐带血来源的外泌体及其应用 Download PDFInfo
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Abstract
本发明涉及一种CD73修饰的人脐带血间充质干细胞来源的外泌体,其通过调节小胶质细胞M1/M2的平衡,在制造用于治疗脊髓损伤的制剂中的应用。CD73修饰的人脐带血间充质干细胞来源的外泌体体内外均可以明显降低ATP和AMP的水平,升高腺苷的水平,分别使用腺苷A2a和A2b受体抑制剂后,间接发现当加入A2b的抑制剂后cAMP显著下降,且PKA明显降低,即进一步间接证明了腺苷是通过A2b受体靶向发挥作用,通过激活下游cAMP/PKA信号通路抑制炎症因子的释放,从而促进M2小胶质细胞的极化,抑制M1小胶质细胞的极化,从而可以治疗脊髓损伤。
Description
技术领域
本发明涉及一种CD73修饰的人脐带血来源的外泌体及其在制备用于治疗脊髓损伤的制剂中的应用,属于西医新用途领域。
背景技术
脊髓损伤(SCI)已经是人类严重致残的重要原因,是脊柱损伤最严重的并发症,如果不及时处理脊髓功能将长期或者永久损伤。在脊髓损伤的病理生理过程与小胶质细胞极化关系密切,通常脊髓小胶质细胞处于静息状态,即M0状态,起免疫监视的作用,只要当中枢神经系统受到损伤后,小胶质细胞便会迅速极化然后以M1和M2两种功能状态发挥作用,研亢表明,体外小胶质细胞M0可通过脂多糖或者干扰素γ诱导向M1状态极化,也可通过IL-4、IL-10和转化生长因子α(TGF-α)诱导向M2状态极化。M1能释放大量的神经炎症性因子如IL-1β、IL-6、TNF-α、ROS(reactive oxygen species)、NO等,趋化巨噬细胞和淋巴细胞向病灶聚集,加重炎症反应和神经细胞的凋亡,参与神经元迟发性损伤。M2能释放抗炎因子如IL-4、IL-13等,吞噬损伤的神经细胞碎片,促进组织修复和神经元的再生。当脊髓损伤时或者组织细胞缺血损伤时,随之将释放大量的ATP,通过经CD39催化生成一磷酸腺苷(AMP)。CD73作为限速酶,可将5’-AMP催化成腺苷(adenosine),迅速降低ATP在局部的聚集,减轻炎症痛苦,通过对小胶质细胞系BV2研亢发现,当对hucMSC-Ex预处理后,hucMSC-Ex表面CD73表达被抑制,结果发现hucMSC-Ex抑制BV2小胶质细胞向M1极化,促进BV2细胞向M2极化的作用减弱了。如果人为的将hucMSC-Ex表面CD73表达增强是否可以出现促进BV2细胞向M2极化的作用,由于MSC具有多向分化能力,分离培养较方便,较低的免疫排斥反应性,具有免疫调节和抗炎活性,是一种理想的组织损伤修复来源细胞。MSC主要来源于骨髓、脂肪组织和脐带。
有研亢证明,人脐带组织来源MSC(human umbilical cord mesechymal stemcell,hucMSC)具有与骨髓源MSC(hbmMSC)相似的生物学特征和功能,且具有比hbmMSC的增殖能力更高、免疫排斥反应更低及获取途径更方便的特点,有望成为理想的MSC来源,所以通过人为的技术将CD73在人脐带组织来源MSC的外泌体中过表达将会在脊髓损伤中起到抗炎及保护神经的作用。
近年来,脊髓损伤的病例数越来越多,而且脊髓损伤后所造成的后遗症将会带来无法估量的损失,无论的是药物的,手术治疗还是高压氧等一系列保守治疗均无法取得满意的疗效,因此,就急需一种对身体损伤小,同时又能带来满意效果的制剂、治疗方法。
发明内容
本发明的目的在于提供一种CD73修饰的人脐带血间充质干细胞来源的外泌体及其用途。
在本发明的第一方面,提供一种CD73修饰的人脐带血间充质干细胞来源的外泌体,其特征在于,所述外泌体被CD73修饰,所述CD73在外泌体上高度表达。
在一个优选例中,所述CD73修饰的人脐带血间充质干细胞来源的外泌体与腺苷A2b受体结合。
在另一个优选例中,所述CD73修饰的人脐带血间充质干细胞来源的外泌体激活下游cAMP/PKA信号通路。
在本发明的另一方面,提供一种药物制剂,包含治疗有效量的被CD73修饰的人脐带血间充质干细胞来源的外泌体以及药学上可接受的辅料,所述辅料包括常规的药用载体以及稀释剂,所述CD73在外泌体上高度表达。
在一个优选例中,所述药物制剂为注射剂。
在另一个优选例中,所述药物制剂每日1次给药,所述药物制剂的每次给药量以CD73修饰的人脐带血间充质干细胞来源的外泌体的质量计为8-40mg。
在本发明的另一方面,本发明提供一种CD73修饰的人脐带血间充质干细胞来源的外泌体在治疗脊髓损伤用制剂的制造中的应用。
在本发明的一个优选例中,所述的CD73修饰的人脐带血间充质干细胞来源的外泌体通过与腺苷A2b受体的结合,抑制ATP,抑制AMP,抑制细胞发生氧化磷酸化。
在本发明的一个优选例中,所述的CD73修饰的人脐带血间充质干细胞来源的外泌体与腺苷A2b受体的结合后,抑制ATP和AMP的合成,从而进一步激活下游cAMP/PKA信号通路发挥作用,从而进一步促进M2型小胶质细胞的极化,抑制M2型小胶质细胞极化。
在本发明的一个优选例中,所述的CD73修饰的人脐带血间充质干细胞来源的外泌体抑制AMP及AMP的合成。
在本发明的一个优选例中,所述的CD73修饰的人脐带血间充质干细胞来源的外泌体促进M2型小胶质细胞极化,抑制M1型小胶质细胞极化。
在本发明的另一方面,本发明提供一种CD73修饰的人脐带血间充质干细胞来源的外泌体在制造用于抑制M1小胶质细胞的促炎作用的制剂中的应用。
在本发明的另一方面,本发明提供一种CD73修饰的人脐带血间充质干细胞来源的外泌体在制造促进M2型小胶质细胞极化用制剂中的应用。
本发明的其他方面由于本文的公开内容,对本领域技术人员而言是显而易见的。
本发明的有益效果
本发明人研亢结果表明,在体内外,CD73修饰人脐带血间充质干细胞来源的外泌体,可以明显降低细胞外ATP、AMP水平,升高腺苷浓度;同时可明显抑制M1型小胶质细胞极化,促进M2型小胶质细胞的极化,后进一步激活下游cAMP/PKA信号通路,抑制下游炎症因袭的释放来诱导抑制作用,CD73修饰人脐带血间充质干细胞来源的外泌体可以用于治疗脊髓损伤。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据本发明这些附图获得其他的附图。
图1为hucMSCs衍生的外泌体和CD73修饰的hucMSCs衍生外泌体所表达的特性的结果。其中,
A显微镜下hucMSCs衍生的外泌体和CD73修饰的hucMSCs衍生外泌体的代表性图像。
B透射电镜下观察hucMSC-Exs和CD73+hucMSCs-Exs的代表性图像。
C采用纳米颗粒跟踪分析(NTA,ZetaView,Particle Metrix Inc.,德国)外泌体的大小分布。
D检测所示蛋白的Western-blotting分析,包括外泌体阳性生物标志物CD73修饰蛋白、CD9和CD63以及外泌体阴性生物标志物calnexin。
E体外测定指示剂量的hucMSC-Exs或CD73+hucMSC-Exs加入100个μM APCP时的对于ATP和AMP水解活性(n=3)。
F体外测定指示剂量的hucMSC-Exs或CD73+hucMSC-Exs不加入100个μM APCP时的对于ATP和AMP水解活性(n=3)。
图2为CD73+hucMSC-Exs在体外促进M2小胶质细胞极化,抑制M1小胶质细胞极化的作用结果。其中,
A采用免疫荧光法测定30ng/ml CD73、30μg/ml hucMSC-Exs和30μg/ml CD73+hucMSC-Exs在加或不加100μM APCP对1μg/ml LPS处理的BV2细胞M1亚群变化的影响。
B用免疫荧光法测定30ng/ml CD73、30μg/ml hucMSC-Exs和30μg/ml CD73+hucMSC-Exs加或不加100μM APCP对5μg/ml IL-4处理的BV2细胞M2亚群变化的影响。
图3为CD73+hucMSC-Exs通过A2b腺苷受体的激活增强M2/M1极化的结果。其中,
A和B在有无在30μg/ml CD73+hucMSC-EXs的情况下,用1μg/ml LPS处理BV2细胞,同时使用1μM MRS1706(A2bR抑制剂)或1μM SCH58261(A2aR抑制剂)。
A刺激10分钟后测定细胞内cAMP水平。
B检测PKA蛋白当A2bR抑制剂MRS1706存在时在BV2细胞中的表达。
C检测iNOS当A2bR抑制剂MRS1706存在时在BV2细胞中的表达
D检测精氨酸酶-1当A2bR抑制剂MRS1706存在时在BV2细胞中的表达
E检测iNOS当A2aR抑制剂SCH58261存在时在BV2细胞中的表达
F为检测精氨酸酶-1(Arginase 1)当A2aR抑制剂SCH58261存在时在BV2细胞中的表达
G为实验组,对照组以及不同浓度的A2bR抑制剂MRS1706存在时其M1小胶质细胞的的mRNA的表达
H为实验组,对照组以及不同浓度的A2bR抑制剂MRS1706存在时其M2小胶质细胞的的mRNA的表达
图4为A2bR相关基因敲低细胞和PKA抑制剂都逆CD73+hucMSC-EXs的作用的结果。其中,
A刺激10分钟后测定细胞内cAMP水平(n=5)。
B刺激10分钟后测定细胞内cAMP水平(n=5)。
C iNOS/Arg-1蛋白在BV2细胞中的表达。
D iNOS/Arg-1蛋白在BV2细胞中的表达。
E mRNA相对表达水平。
F mRNA相对表达水平。
G iNOS/Arg-1蛋白在BV2细胞中的表达情况,
H iNOS/Arg-1蛋白在BV2细胞中的表达情况。
图5为CD73+hucMSC-Exs通过调节小胶质细胞M1/M2极化,改善小鼠SCI,降低细胞内cAMP水平的结果。其中,
A为脊髓损伤造模后不同时间运动功能BBB评分,
B为脊髓损伤后不同时间脑脊液中cAMP水平,
C为组织学图像(H&E染色),
D为损伤脊髓纵切面Nissl染色,
E和F显示了小胶质细胞/巨噬细胞亚群的流式细胞仪代表性点迹分布,分别以CD206和CD86作为M2和M1小胶质细胞的生物标志物,数据计算为M2∶M1的比率。
图6为CD73+hucMSC-Exs对小胶质细胞的作用机制的示意图。
具体实施方式
在本发明中,建立了CD73过表达的工程外泌体作为纳米药物载体,并公开了CD73+hucMSC-EXs治疗可以减轻小鼠脊髓损伤后的炎症反应,在体外调节巨噬细胞/小胶质细胞M2∶M1的极化。CD73+hucMSC-EXs降低APT浓度,促进腺苷水平,进一步激活A2bR和cAMP/PKA信号通路。因此,CD73+hucMSC-EXs工程外泌体在SCI中具有抗炎作用。
众所周知,炎症在SCI的发病机制中起着重要的作用。炎症和组织损伤可产生过多的系统性ATP进入细胞外空间,并触发继发性损伤的级联反应。细胞外ATP可被CD39和CD7335的膜结合酶两步降解为免疫抑制腺苷,这在急性炎症中是至关重要的。CD73是AMP转化为腺苷的最后一步的限速酶。有报道称,BV2细胞中过表达的CD73通过介导巨噬细胞/小胶质细胞极化发挥神经保护作用。然而,这种酶能否作为一种药物来调节腺苷的水平从未被研亢。值得注意的是,CD73是一种新兴的免疫检查点和癌症治疗的理想靶点。换句话说,CD73过表达可能促进癌的进展和复发。因此,CD73强烈的免疫抑制可能会限制其作为全身药物的应用,但使用纳米载体对CD73进行局部传递可能是一种替代方法。
外泌体可被用来运输遗传物质或药物到靶细胞。外泌体是纳米大小的脂双层膜包裹的小泡,这使它们能够渗透血脑屏障。它被开发用于治疗各种疾病,包括癌症和中枢神经系统紊乱。此外,msc-来源的外泌体(MSC-EXs)具有msc的原始特性,如再生和损伤减轻作用。与间充质干细胞相比,MSC-EXs具有更大的稳定性和可操作性、更低的免疫排斥反应几率和无恶性转化风险等不同优势。MSC-EXs通过促进血管生成和轴突生长,调节炎症和免疫反应,在SCI修复中发挥关键作用。
因此,本发明中选择hucmc-exs作为CD73的纳米载体。
本发明中,通过慢病毒转导获得CD73+humscs,然后分离出CD73+humscs-exs作为纳米药物。结果表明,CD73+hucMSC-EXs对ATP和AMP具有剂量依赖性水解作用,所产生的腺苷与A2bR结合。腺苷受体分为A1R、A2aR、A2bR和A3R,它们通过生物信号转导产生不同的生理效应。A1R和A3R与Gi偶联,抑制cAMP水平,而A2aR和A2bR与刺激性Galpha蛋白(Gs)偶联,增加cAMP43水平。
A2aR对于巨噬细胞来说,A2bR的激活对于腺苷刺激IL-10的产生和抑制一氧化氮的释放是至关重要的。此外,研亢表明A2bR激活可通过腺苷刺激替代M2/M1极化。因此,本发明中,使用A2aR、A2bR和A2bR敲低细胞的选择性拮抗剂,证明A2bR负责CD73+hucMSC-EXs水解腺苷刺激的小胶质细胞选择性活化。
A2bR激活后,cAMP水平升高。蛋白激酶A(PKA)是cAMP的胞内受体。在发明亢中,发现A2bR信号通路通过三种方法增加cAMP/PKA的表达,包括A2bR拮抗剂、A2bR敲低和PKA抑制剂。其他研亢也表明A2bR激活可诱导cAMP/PKA信号通路。因此,活化的PKA抑制了M1的活化,增加了M2的极化。因此,级联的促炎细胞因子被下调,如TNF-TNF-TNF-、IL-1抑制因子和IL-6;而抗炎细胞因子上调,如IL-10和IL-4。
本发明中,CD73在人脐带血间充质干细胞来源的外泌体上高度表达。本发明中,高度表达是指:一般情况下外泌体仅表达CD63和CD81,即让外泌体改造后可以检测到CD73表达即为高表达。
本发明中,CD73的DNA序列如下(即序列表中的SEQ ID NO:1):
本发明的CD73在人脐带血间充质干细胞来源的外泌体的制造方法如下:
在无菌条件下,清洗并取出脐带血。将其切成1mm片,转入DMEM中;培养。离心,洗涤,去除不贴壁细胞。细胞以1×106/mL的密度装于培养瓶中,每3天更换一次培养液。细胞达到90%的融合,出现成纤维细胞样细胞集落,用胰蛋白酶消化,进入新的培养皿。在干细胞培养基中培养humscs进一步扩增。BV2细胞是一种小胶质细胞,在完全培养基和胎牛血清和青霉素链霉素抗生素下培养。
使用脂质转染人胚胎肾细胞系和包装质粒。将humscs制备于12孔板中,培养至70-80%融合。第二天,将制备好的humscs用慢病毒转染,并加入10以g/ml的聚乙烯。培养基每12h更换一次。另外,24h时加入羟丙霉素,每2天更换一次。最后,利用荧光显微镜和光镜检查收获的CD73+humscs稳定细胞株的活性。
将上述得到的CD73+ humscs稳定细胞株用PBS洗涤2次后,在无血清的lp-dmem培养液中培养至约80%融合(confluent)48h。收集上清液,采用超离心离心法分离外泌体(MICROCL,Thermo Fisher Scientific,Inc.美国),以清除细胞碎片。使离心后的上层清液收集每次1000g 10分钟,随后在2000g离心10分钟和在3200g离心30分钟。此外,充满了PBS上层清液,然后,再次超速离心100000g两次70分钟。得到人脐带血间充质干细胞来源的外泌体。
作为注射剂时,CD73修饰的人脐带血间充质干细胞来源的外泌体在体内外都被证实可以抑制M1小胶质细胞的极化,促进M2小胶质细胞的极化,激活下游cAMP/PKA信号通路,进一步抑制下游炎症因子的释放,从而发挥神经的抗炎及保护作用。
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清楚、完整地描述,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。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验例
CD73+hucMSC-Exs对C57BL/6小鼠脊髓损伤具有抗炎及保护作用的实验实验材料
采用生理盐水将CD73+hucMSC-Exs稀释成不同浓度的药物溶液。
6周龄雄性ICR小鼠(20-25g)购自中国上海SLAC公司,饲养于长海医院SPF级动物房。
BV2细胞由海军军医大学提供,其他材料在市场上购买,无特殊要求。
实验方法:
实验例1.细胞培养
脐带组织取自知情同意的健康母亲。在无菌条件下,脐带血被清洗并取出。将其切成1mm片,转入含有10%胎牛血清、5%HS、1%P/S(v/v)和胶原酶II(1g/L)的DMEM中;(南京科根生物科技有限公司,中国南京),37℃,5%CO2培养箱。离心,用磷酸盐平衡生理盐水(PBS)洗涤,去除不贴壁细胞。细胞以1×106/mL的密度装于培养瓶中,每3天更换一次培养液。10天后,细胞达到90%的融合,出现成纤维细胞样细胞集落,用胰蛋白酶消化,进入新的培养皿。在干细胞培养基(Cyagen,中国广州)中培养humscs进一步扩增。BV2细胞是一种小胶质细胞,在完全培养基(DMEM,Gibco,Carlsbad,CA,USA)和10%胎牛血清(FBS,Gibco,Carlsbad,CA,USA)和1%青霉素链霉素抗生素(HyClone)下培养。在上述培养基中培养的细胞定义为M0巨噬细胞。LPS/白介素4(IL-4)作用于M0巨噬细胞8小时,激活M1/M2阶段。
实验例2.慢病毒载体的产生和humscs的转导
为了制造慢病毒载体,pCDH慢病毒质粒被用于在二级生物安全实验室制造病毒。pCDH-GFP载体基于CD73过表达质粒构建,该质粒由包含CD73序列的合成基因制备。使用Lipofectamine 3000转染人胚胎肾293T(HEK-293T)细胞系和包装质粒(Thermo FisherScientific,Waltham,MA)。以嘌呤霉素(puromycin)处理humscs,确定最低致死浓度。在>20°l/ml浓度下持续6天,几乎100%的humscs凋亡;因此,选择20个丙烯l/ml作为普罗霉素的最佳筛选浓度。将humscs制备于12孔板中,每孔3-5×105个细胞,培养至70-80%融合。第二天,将制备好的humscs用慢病毒转染,并加入10μg/ml的聚乙烯(Sigma,德国)。培养基每12h更换一次。另外,24h时加入20个羟丙霉素,每2天更换一次。最后,利用荧光显微镜和光镜检查收获的CD73+humscs稳定细胞株的活性。
实验例3.外泌体的分离和鉴定
将上述实验例2所得到的CD73+ humscs稳定细胞株用PBS洗涤2次后,在无血清的lp-dmem培养液(Thermo Fisher Scientific,Inc.USA)中培养至约80%融合(confluent)48小时。收集上清液,采用超离心离心法分离外泌体(MICROCL,Thermo FisherScientific,Inc.USA),以清除细胞碎片。使离心后的上层清液收集每次1000g 10分钟,随后在2000g离心10分钟和在3200g离心30分钟。此外,充满了PBS上层清液,然后,再次超速离心(altracentrifugated)100000g两次70分钟。最后,上层清液是透过0.22μm孔隙过滤器(微孔)和液纯化和收集。外泌体的鉴定,通过透射电镜(TEM,JEM-2100F,JapanElectronics Co.,Ltd.)鉴定纯化后的外泌体的形态。采用纳米颗粒跟踪分析(NTA,ZetaView,Particle Metrix Inc.德国)检测颗粒的粒径分布和浓度。
实验例4.外泌体ATP和AMP水解的测定
外泌体用超离心法反丸,用MES缓冲液(Sigma)重悬以去除PBS中的无机磷酸盐。然后,给予20个三磷酸腺苷或安培酸(Sigma),每500°l的MES治疗1小时,指示剂量为3、10或30°g/ml的hucMSCs-EXs或CD73+hucMSCs-EXs,并添加或不添加100个ampcp(Santa CruzBiotechnology,Santa Cruz,CA)。ATP测定试剂盒(Beyotime,中国上海)测定ATP浓度,SensoLyte MG磷酸盐测定试剂盒(Anaspec,美国)测定ATP或AMP产生的磷酸盐,腺苷测定试剂盒(BioVision,CA,美国)测定腺苷浓度。
实验例5.免疫荧光
0.1%Triton-100透化5min,5%BSA封闭,抗inos抗体1∶100孵育过夜,进行荧光显微镜观察。Abcam,ab49999)和抗arg1抗体(1∶100,Abcam,ab133543)。用二抗Alexa Fluor488山羊抗小鼠IgG(H+L)抗体/Alexa Fluor 594马抗兔二抗孵育细胞(均为1∶1000;杰克逊免疫研亢,韦斯特格罗夫,宾夕法尼亚州)在室温下1小时。使用DAPI溶液进行核染色5min。使用徕卡TCA SP8共焦激光扫描显微镜(徕卡,德国)拍摄图像。
实验例6.免疫印迹
细胞和外泌体用冷冻放射免疫沉淀试验缓冲液(Solarbio,中国,北京)和蛋白酶和磷酸酶抑制剂混合物(Abcam,剑桥,MA,美国)裂解,用BCA蛋白测定试剂盒(Beyotime,P0010)测定蛋白浓度。然后将蛋白样品溶解于sds-聚丙烯酰胺凝胶电泳,并转移到硝酸纤维素膜上。在锁定缓冲液(TBST)中被阻断,与一抗(1∶1000)在4℃孵育,过夜21,22。CD73、CD9、CD63、钙连蛋白(calnexin)、PKA、iNOS、和Arg1抗体购自Abcam(Abcam,CA,USA)。GAPDH抗体购自Santa Cruz Biotechnology(Santa Cruz,CA,USA)。用TBST洗涤三次后,膜与辣根过氧化物酶偶联二抗(1∶50 000)孵育,增强化学发光形成条带。使用Image J(NIH Image J系统,Bethesda,MD)进行图像分析。
实验例7.实时定量PCR(qRT-PCR)分析
按照制造商的说明使用TRIzol试剂(Thermo Fisher Scientific,USA)分离总RNA。RNA浓度由NanoDrop ND-2000(Thermo Fisher Scientific,USA)定量。采用SYBRGreen试剂进行qRT-PCR mRNA定量。相应引物列于补充表1。
补充表1实时PCR引物的序列Sequence of the real-time PCR primers
实验例8.细胞内营的测量
细胞内cAMP使用Biotrak(EIA)特异性酶免疫测定系统(Amersham),按照制造商的说明操作。简单地说,105个BV2细胞被镀膜,并用11M/mL的LPS孵育。在450nm处读取光密度并计算结果。
实验例9.转染的成分
BV2细胞(15,000个细胞/ml)被沉积24小时,然后分别与108个shRNA慢病毒转导颗粒(用于人A2b ADO受体)和混乱的非靶向shRNA(SHC002V)作为对照孵育。按照厂家建议转染细胞,然后以2μg/ml加入嘌呤霉素(Sigma公司)选择细胞。通过定量PCR检测A2bR的表达,验证其knockdown的效果。
实验例10.脊髓损伤动物模型
6周龄雄性ICR小鼠(20-25g)购自中国上海SLAC公司。所有小鼠均在SPF(specificpathogen free,SPF)实验室饲养,实验程序经机构实验动物护理与使用委员会批准。用戊巴比妥钠(50mg/kgi.p.)麻醉小鼠。双侧椎板切除T8-T9显露脊髓。使用纽约大学的撞击器在6.25毫米的高度使用10克的杆来造成重降损伤。损伤后,关闭肌肉和皮肤,并将小鼠置于温度和湿度可控的室内。每日3次进行人工膀胱排空。
实验例11.SCI中hucMSC衍生外泌体的治疗
假手术组(n=15)行双侧椎板切除术,未损伤脊髓,并在创伤处注射生理盐水。此外,SCI老鼠被随机分为四组,每组15只老鼠分别用生理盐水治疗组(SCI),20μg hucMSC-EXs(SCI+hucMSC-EXs组)或20μg CD73+hucMSC-EXs(SCI+CD73+hucMSC-EXs集团),或20ngrmCD73(重组鼠标CD73,北京拜尔赖博科技有限公司有限公司,北京,中国),注射在伤口一天一次,10天受伤。
实验例12.行为测试
采用BBB评分(Basso-Beattie-Bresnahan,BBB)评分,由两名训练有素、不考虑实验动物分组的观察人员对损伤后1、3、7、14、21、28、35和42天后肢运动功能恢复情况进行评价。在这些测试中,老鼠被允许在开阔的田野中自由活动4分钟,然后计算并记录它们的平均运动能力得分。
实验例13.脑脊液中ATP的测定
用异氟醚和氧麻醉小鼠,用玻璃毛细管直接刺穿大池,如前所述。收集CSF,使用ATP测定试剂盒(Beyotime,中国上海)测定ATP。
实验例14.H&E染色和Nissl染色
动物被安乐死,并向左心室灌注4%多聚甲醛,如之前描述的[13,14]。整个脊髓被迅速切除并包埋在石蜡中。以损伤中心为中心的1厘米长的脊髓在5纵米处制作纵切面。各脊髓切片分别以苏木精、伊红染色(H&E)和尼氏染色。
实验例15.流式细胞术和细胞因子分析
每天将humsc衍生的外泌体注射到小鼠伤口中,并3d执行以获取组织,如前文所述。这些动物被深度麻醉,血液通过心脏穿刺抽走。以T11为中心的一段10毫米的线段被取出,并迅速冷冻在乙醇干冰浴中。为了分离小胶质细胞,脊髓组织被移除并在冰-coldDulbecco改良Eagle培养基(DMEM)(Invitrogen公司)中用剪刀切碎。随后,用0.25%胰蛋白酶(Invitrogen公司)在37℃水浴中消化30分钟形成单眼悬浮液,然后按照制造商的说明在室温下对CD206(M2小胶质细胞生物标志物)和CD86(M1小胶质细胞生物标志物)共染色45分钟。样品采用FACSAria III流式细胞仪(BD Biosciences,San Jose,CA,USA)检测,用FlowJo软件v.7.6.1(https://flowjo.com/)分析。此外,Bio-plex系统(Bio-Rad,CA)和23倍细胞因子阵列试剂盒(Bio-Rad)用于脊髓标本的细胞因子分析。
实施例1.humc-exs和CD73+humc-exs的分离与鉴定
首先,在光镜下观察根据实验例1的方法从脐带组织中收获的humscs和根据实验例2、3的方法得到的经慢病毒转导后的CD73+humscs,见图1A。分离后的humsc-exs和CD73+humsc-exs通过透射电镜进行形貌鉴定,典型结构如图1B所示。图1C用NTA检查粒径分布和浓度。hucmc-exs的直径为103.41±42.03nm,CD73+hucmc-exs的直径为106.51±53.99nm。BCA试剂盒测定外泌体蛋白浓度为2.5mol/mol。此外,用Western blot检测阳性标记(CD73、CD9和CD63)和阴性标记(钙连蛋白),见图1D。CD9和CD63在外泌体中保存并检测到,钙连蛋白存在于细胞内质网中,而不在外泌体中。它们被广泛用作检测外泌体的生物标记物。结果表明,从humscs中分离出的胞外囊泡是真正的外泌体,CD73在CD73+humscs和CD73+humcs-exs中表达明显增高。
实施例2.CD73+hucMCSs-EXs在体外对ATP水解成腺苷和促进小胶质细胞M1向M2表型极化的作用
根据实验例4的方法,即通过APT/cAMP-PKA信号通路,并对其腺苷受体A2b进行抑制,检测其下游PKA的情况,间接检测了ATP和AMP的水解活性。结果表明,hucMCSs-EXs和CD73+hucMCSs-EXs水解ATP和AMP的作用剂量依赖,而CD73特异性抑制剂APCP显著降低了这一作用(图1E和F、)。此外,CD73+hucMCSs-EXs的水解作用明显强于hucMCSs-EXs。通过调节小胶质细胞M1/M2平衡,减少神经炎症因子的释放,对中枢神经的保护具有深远意义。本研亢将小鼠BV2细胞分离培养7天,分别用1mol/ml LPS或5mol/ml IL-4诱导细胞向M1或M2细胞极化。然后选择iNOS和精氨酸酶1作为M1/M2细胞的表面标记物(图2)。最后显示,humsc-exs和CD73+humsc-exs,CD73可以降低LPS诱导的iNOS的表达。CD73+hucMSC-EXs降低最大,而APCP抑制其作用。另一方面,也显示CD73+hucMSC-EXs显著增加精氨酸酶1的表达,而APCP则逆转了这一现象。
实施例3.CD73+hucMSC-EXs通过激活A2b腺苷受体增强M2极化和缓解M1极化
为了验证CD73过表达的下游靶点,分别使用SCH58261和MRS1706作为A2a和A2b受体的抑制剂。CD73+hucMSC-Exs通过A2b腺苷受体的激活增强M2/M1极化。在有无在30μg/mlCD73+hucMSC-EXs的情况下,用1μg/ml LPS处理BV2细胞,同时使用1μM MRS1706(A2bR抑制剂)或1μM SCH58261(A2aR抑制剂)已知A2Rs介导小胶质细胞炎症,A2Rs是gs连接蛋白,增加cAMP。在图3A中,30mol/1 CD73+hucMCSs-EXs显著增加cAMP水平。MRS1706(A2bR抑制剂)显著降低,而SCH58261(A2aR抑制剂)无显著降低。对于cAMP的下游靶点,CD73+hucMCSs-EXs处理后PKA也有所升高,MRS1706对PKA有明显抑制作用,而SCH58261对PKA无明显抑制作用(图3b)。此外,精氨酸酶1和iNOS常被用作小胶质细胞亚型的标记,因为精氨酸酶1可以有效地下调iNOS引起的一氧化氮生成,激活抗炎功能。LPS处理后,CD73+hucMCSs-EXs显著降低iNOS水平;而IL-4处理后,CD73+hucMCSs-EXs显著提高精氨酸酶1的水平。
MRS1706对CD73+hucMCSs-EXs的抑制作用呈剂量依赖性(图4C和4D)。相比之下,SCH58261对CD73+hucMCSs-EXs的影响不大(图4E和4F)。此外,检测M1 mRNA标记物(TNF-inhibitor、IL-1inhibitor、iNOS和CD86)的表达和M2 mRNA标记物(精氨酸酶、IL-10和CD206)的表达。结果与western blot结果相似(图4G和图4H)。因此,CD73+hucMCSs-EXs可能通过激活A2b腺甘酸受体增强M2极化和中度M1极化,而不是A2a腺甘酸受体。
实施例4.A2bR敲低细胞和PKA抑制剂逆转CD73+hucMSC-EXs对交替激活的小胶质细胞的刺激作用
实验过程如上述实验例9所示。A2bR通过永久转染A2bR shRNA敲除。经LPS和CD73+hucMCSs-EXs处理后,shRNAA2bR组的cAMP浓度较shRNA打乱组显著降低(图4A)。
其中,图4(A-F)为BV2细胞永久转染,通过shRNA或A2bR shRNA进行沉默。然后,在有无30μg/ml CD73+hucMSC-Exs的情况下,用1μg/ml LPS或5μg/ml IL-4处理细胞。所得到的结果的图。
CD73+hucMCSs-EXs处理A2bR shRNA组时,PKA的标准化表达水平也显著降低(图4B)。此外,在M1/M2亚型中,CD73+hucMCSs-EXs下调了shRNA打乱组和shRNAA2bR组LPS诱导的iNOS水平,但shRNA A2bR组的iNOS水平显著高于shRNA打乱组。CD73+hucMCSs-EXs和IL-4处理shRNA A2bR组的精氨酸酶1水平明显低于shRNA打乱组(图4C和4D)。同样,在A2bR敲除细胞中观察到M1/M2标记的mRNA,其结果与Western blot结果一致(图4E和4F)。结果证实了A2bR在CD73+hucMCSs-EXs的作用中起重要作用。同时,cAMP信号通过细胞内PKA的激活进行传递,使用PKA抑制剂H-89来确定这个下游靶点。说明在LPS诱导后,iNOS水平升高。CD73+hucMCSs-EXs降低了iNOS的表达,而H-89抑制了iNOS的表达。H-89降低了CD73+hucMCSs-EXs对精氨酸酶1水平的上调作用(图4G和4G)。因此,A2bR被确定为CD73+hucMCSs-EXs产生的腺苷受体,下游生物标志物可能通过A2bR/cAMP/PKA信号通路被激活。
实施例5.CD73+hucMSC-EXs改善小鼠SCI,降低细胞内cAMP水平
采用Allen方法建立小鼠脊髓损伤模型。损伤后10天分别给予生理盐水、hucMSC-EXs、CD73+hucMSC-EXs(SCI+CD73+hucMSC-EXs组)、重组小鼠CD73治疗。首先,为了比较五组小鼠的不同运动功能水平,记录了术后1、3、7、14、21、28、35、42天不同时间的BBB评分。根据评分标准计算练习分数,用Excel记录并绘制不同分组BBB分数的标线。实验过程如上述实验例12以及实验例4所示。
标线显示SCI+CD73+hucMCSs-EXs组在SCI后运动功能障碍方面明显优于SCI组。SCI+hucMCSs-EXs组也优于SCI组和SCI+CD73组(图5A)。提取小鼠脑脊液,检测病理条件下1、3、7、14、21、28、35、42天ATP浓度。假手术组的浓度基本不变。SCI+CD73+hucMCSs-EXs组ATP水平明显高于SCI组。SCI+hucMCSs-EXs组ATP水平中度升高。ATP水平在1-7天内上升,而在第14天后由于第10天停止治疗而减慢(图5B)。
此外,选择小鼠模型段脊髓组织在损伤后7周进行HE染色和Nissl染色。SCI组表现出尼氏体神经元紊乱、肿胀和空泡结构。HucMCSs-EXs减少了损伤,CD73+HucMCSs-EXs表现更好(图5C和5D)。
实施例6.CD73+hucMSC-EXs在小鼠SCI后调节M1/M2极化和促炎细胞因子
在SCI后3天的小鼠上使用流式细胞仪测定小鼠脊髓样本中M2和M1小胶质细胞的比例。选择CD206和CD86作为M2和M1小胶质细胞的生物标志物。实验过程如上述实验例15所示。
结果表明,SCI+CD73+hucMCSs-EXs组计算出的M2:M1明显高于其他组(图6E和6F)。另外,在治疗后3天,用bio-plex系统定量分析SCI小鼠脊柱标本中的细胞因子。将各组细胞因子含量标准化后,建立各组的热图。SCI+CD73+hucMCSs-EXs组中,IL-1细胞因子、IL-6、TNF-a、MCP-1、IFN-a和MIP-1细胞因子显著降低。SCI+CD73+hucMCSs-EXs组抗炎细胞因子IL-4、IL-10明显升高。这样,CD73+hucMCSs-EXs上调了M2:M1的极化,减轻了脊髓损伤修复中的炎症反应。
利用外泌体作为纳米药物载体,当外泌体表面的特定蛋白在未来被设计用于靶向细胞时,副作用可以减少。这些结果均表明,CD73修饰的人脐带血间充质干细胞来源的外泌体可以治疗脊髓损伤。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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Claims (9)
1.一种人脐带血间充质干细胞来源的外泌体,其特征在于,所述外泌体被CD73修饰,所述CD73在外泌体上高度表达。
2.根据权利要求1所述的外泌体,所述CD73修饰的人脐带血间充质干细胞来源的外泌体与腺苷A2b受体结合。
3.根据权利要求1或2所述的外泌体,其特征在于,所述CD73修饰的人脐带血间充质干细胞来源的外泌体激活下游cAMP/PKA信号通路。
4.一种药物制剂,其特征在于,包含治疗有效量的被CD73修饰的人脐带血间充质干细胞来源的外泌体以及药学上可接受的辅料,所述辅料包括药用载体以及稀释剂,所述CD73在外泌体上高度表达。
5.根据权利要求4所述的药物制剂,其特征在于,所述的药物制剂为注射剂。
6.根据权利要求4或5所述的药物制剂,其特征在于,所述药物制剂每日1次给药,所述药物制剂的给药量以CD73修饰的人脐带血间充质干细胞来源的外泌体的质量计为8-40mg。
7.一种权利要求1-3中任一项所述的人脐带血间充质干细胞来源的外泌体在治疗脊髓损伤用制剂的制造中的应用。
8.一种权利要求1-3中任一项所述的人脐带血间充质干细胞来源的外泌体在制造用于抑制M1小胶质细胞的促炎作用的制剂中的应用。
9.一种权利要求1-3中任一项所述的人脐带血间充质干细胞来源的外泌体在制造促进M2型小胶质细胞极化用制剂中的应用。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107217034A (zh) * | 2017-04-20 | 2017-09-29 | 深圳市赛欧细胞生物科技有限公司 | 一种人脐带间充质干细胞源外泌体及其获取方法和应用 |
TW202034932A (zh) * | 2019-03-18 | 2020-10-01 | 佛教慈濟醫療財團法人 | 間質幹細胞之胞外泌體及其用途 |
WO2020215024A1 (en) * | 2019-04-18 | 2020-10-22 | The Regents Of The University Of California | Exosome mimicking nanovesicles making and biological use |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107217034A (zh) * | 2017-04-20 | 2017-09-29 | 深圳市赛欧细胞生物科技有限公司 | 一种人脐带间充质干细胞源外泌体及其获取方法和应用 |
TW202034932A (zh) * | 2019-03-18 | 2020-10-01 | 佛教慈濟醫療財團法人 | 間質幹細胞之胞外泌體及其用途 |
WO2020215024A1 (en) * | 2019-04-18 | 2020-10-22 | The Regents Of The University Of California | Exosome mimicking nanovesicles making and biological use |
Non-Patent Citations (3)
Title |
---|
SHUN XU等: "Ecto-5′-nucleotidase (CD73) attenuates inflammation after spinal cord injury by promoting macrophages/microglia M2 polarization in mice", 《JOURNAL OF NEUROINFLAMMATION》 * |
WEN-ZHAO LIU等: "Mesenchymal stem cell-derived exosomes: therapeutic opportunities and challenges for spinal cord injury", 《STEM CELL RES THER.》 * |
马铭辰等: "间充质干细胞外泌体对组织再生的作用及其机制的研究进展", 《中国医科大学学报》 * |
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