CN117286094A - 一种人类器官来源外泌体的制备方法及其应用 - Google Patents
一种人类器官来源外泌体的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种人类器官来源外泌体的制备方法及其应用。该制备方法，包括下述步骤：1)以人的诱导多能干细胞或组织来源的成体干细胞或分化细胞为起始细胞制备类器官；2)收集类器官的培养基上清和/或组织解离后的类器官细胞悬液上清，通过超速离心从类器官培养基上清或组织解离后的类器官细胞悬液的上清中分离外泌体。本发明获取的外泌体来源于正常类器官，非肿瘤类器官，外泌体组成成分和多样性与正常人体更为一致；本发明从类器官解离液中提取外泌体，可获得类器官细胞间隙内的外泌体，与培养上清中获得的外泌体互为补充，得到更接近于人体器官内的外泌体成分。
Description
技术领域
本发明属于生物医药领域，具体涉及一种人类器官来源外泌体的制备方法及其应用。
背景技术
外泌体(Exosomes，EVs)是一种直径在30-200nm的微小囊泡，富含脂质、蛋白质/多肽、mRNA、microRNA(miRNA)、长链非编码RNA以及完整的细胞器等多种成分。外泌体频繁穿梭于细胞之间，参与了机体中许多重要的生理生化过程，如细胞通信、迁移、血管新生、自身免疫反应、组织损伤的修复等。
1983年，外泌体首次在绵羊网织红细胞中被发现，起初认为是细胞排泄物，1987年Johnstone命名为“exosome”。近40年来，随着研究的深入，发现外泌体广泛存在于人体尿液、血浆、唾液、母乳、脑脊液、羊水、腹腔积液等多种不同体液中。目前多使用体液或细胞培养上清作为提取外泌体的原材料，采用超高速离心的方法获取；而组织样本来源的EVs相比于细胞系培养或者从体液中分离的EVs具有明显的优势，包含更丰富的信息来源，是研究外泌体的重要领域，但由于组织来源外泌体获取难度较大，相关研究有限。
发明内容
为了研究组织来源的EVs，本发明提供了一种人类器官来源外泌体的制备方法。
为实现上述目的，本发明是通过以下技术方案实现的：
一种人类器官来源外泌体的制备方法，包括下述步骤：
1)以人的诱导多能干细胞或组织来源的成体干细胞或成体分化细胞为起始细胞制备类器官；
2)收集类器官的培养基上清和/或组织解离后的类器官细胞悬液上清，通过超速离心从类器官培养基上清或组织解离后的类器官细胞悬液的上清中分离外泌体。
本发明中，所述类器官是指利用多能干细胞或成体干细胞或成体分化细胞，在体外，通过三维培养方式形成具有来源器官关键结构和功能的组织类似物。
所述类器官作为人体的衍生物，含有器官特有的多种细胞类型、结构特征以及部分功能特性，与人体器官拥有高度相似的组织学和基因型特征。
上述方法步骤1)中，我们选用人类器官作为外泌体来源的组织，包括皮肤1、脑2、肺3、肾4、心脏5等，以人的诱导多能干细胞或组织来源的成体干细胞或成体分化细胞为起始细胞，在相应信号通路的调控下1-5，通过逐步添加细胞因子(如FGF2、Activin A、BMP4、VEGF-A、FGF7、FGF10、EGF、HGF等)或小分子化合物组合(如SB431542、LDN-193189、LY294002、CHIR99021、Dorsomorphin、Dexamethasone、IBMX、A83-01等)，分别分化为皮肤、脑、肺、肾、心脏等类器官(图1)。
上述方法步骤2)中，所述组织解离后的类器官细胞悬液上清的具体制备方法如下：DPBS(杜氏磷酸盐缓冲液)清洗类器官(如可清洗两次)，使用TrypLETMExpress酶于37℃培养箱中消化类器官10-30min，收集细胞悬液于1000-2000r.p.m转速离心5-10min去除细胞，转速3000-5000g离心10-20min去除死细胞，取上清用于后续实验；酶消化的目的是使组织EVs扩散到培养基中。
上述方法步骤2)中，所述超速离心的具体方法为：
先低速离心，取上清后超高速离心，弃上清，用1×PBS重悬沉淀后使用0.22μm/0.45μm滤膜过滤，以弃掉上清中的杂质，再次超高速离心，最终获得的外泌体直径在50-300nm范围内(图2)。
其中，所述低速离心的条件为：2000-5000g，离心10min，取上清；再10000g，离心30min，离心的温度为4℃。
所述超高速离心的条件为：离心转速为100000g-120000g，离心时间为60-90min，离心的温度为4℃。
上述方法制备得到的外泌体也属于本发明的保护范围。
本发明还保护上述外泌体的应用。
所述应用是其在制备用于损伤组织修复的产品中的应用。
外泌体的用途：不同细胞分泌的外泌体具有不用的组成成分和功能，干细胞来源的外泌体与干细胞具有相似的生物学性能，如促进损伤组织的再生修复。以皮肤类器官为例，皮肤类器官本身就可以促进皮肤创面的修复以及附属器的再生，皮肤类器官来源的外泌体用于皮肤创面的修复，即避免了干细胞治疗的免疫排斥、伦理问题等缺点，且具有高稳定性、易于储存、无需增殖、便于定量使用等优势，与单一的细胞因子比较，具有较高的安全性和更大的组织再生潜能。
与现有技术相比，本发明具有如下有益效果：
本发明获取的外泌体来源于正常类器官，非肿瘤类器官，外泌体组成成分和多样性与正常人体更为一致；本发明从类器官解离液中提取外泌体，可获得类器官细胞间隙内的外泌体，与培养上清中获得的外泌体互为补充，得到更接近于人体器官内的外泌体成分。
附图说明
图1为源于人诱导多能干细胞的带有毛囊等附属器的类器官的形态图，图中，从左至右依次为：人皮肤、脑、肺、肾、心脏、肝脏的形态图；
图2为透射电镜观察类器官来源外泌体，图中，从左至右依次为：人皮肤、脑、肺类器官来源的外泌体；
图3为皮肤类器官来源外泌体粒径分析(Nanoparticle Tracking Analysis，NTA)检测结果。
图4为WB检测类器官来源外泌体标志蛋白的表达；结果表明人肾(KO)、心脏(CO)和皮肤(SO)类器官提取的外泌体均可成功检测到标志蛋白CD63，而不表达外泌体的阴性标志物Calnexin，而皮肤类器官表达阳性标志物CD81。
图5类器官来源外泌体NTA检测结果；图中，左侧为肾类器官来源外泌体，右侧为心脏类器官来源外泌体。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
实施例1、制备人类器官(皮肤)来源外泌体
1)本实施例选用人皮肤类器官作为外泌体来源的组织，根据Lee等人建立的皮肤类器官诱导方法建立皮肤类器官1。以人的诱导多能干细胞为起始细胞，在相应信号通路的调控下，通过逐步添加细胞因子和小分子化合物组合(第0天添加10μMSB431542、4ng/mLFGF2和2.5ng/mL BMP4，第3天添加200nM LDN-193189和50ng/mL FGF)，分化为皮肤类器官(见图1)。
2)对含皮肤类器官的培养基进行离心，分别收集皮肤类器官的培养基上清以及沉淀(类器官)，并对类器官沉淀进行组织解离得到组织解离后的类器官细胞悬液的上清，收集好两种来源的上清后，通过超速离心从类器官细胞悬液上清或培养基上清中分离外泌体。
其中，组织解离后的类器官细胞悬液上清的制备方法如下：DPBS(杜氏磷酸盐缓冲液)清洗类器官两次，使用5mL TrypLETMExpress酶于37℃培养箱中消化类器官30min，收集细胞悬液于1000r.p.m转速离心5min去除细胞，转速4000g离心10min去除死细胞，取上清用于后续实验。
上述超速离心的具体方法为：先4℃低速(2000g，离心10min，取上清；10000g，离心30min)离心，取上清后超高速离心(4℃，110000g，离心75min)，弃上清，用1×PBS重悬沉淀后使用0.22μm滤膜过滤，以弃掉上清中的杂质，再次超高速离心，离心的转速为110000g，离心时间为75min，离心的温度为4℃。最终通过外泌体粒子检测(NTA)，我们获得的人皮肤类器官来源外泌体的粒径大小为159.5nm(图3)。WB检测结果显示皮肤类器官来源外泌体表达外泌体阳性标志物CD63和CD81，不表达阴性标志物Calnexin(图4)。
实施例2、制备人类器官(肾/心脏)来源外泌体
参照实施例1的制备方法，将步骤1)中的皮肤类器官替换为其它人类器官(如肾或心脏)作为外泌体来源的组织，获得相应的人类器官(肾/心脏)来源的外泌体。
其中，肾脏类器官的建立方法参照下述文献4中记载的方法；心脏类器官的建立方法参照下述文献5中记载的方法。
WB检测结果显示，类器官(肾/心脏类器官)来源的外泌体不表达阴性标志物Calnexin，表达外泌体阳性标志物CD63和CD81(图4)。NTA检测发现，肾类器官来源的外泌体的粒径大小为156.1nm，心脏类器官来源的外泌体的粒径大小为137.1nm(图5)。
参考文献：
1Lee,J.et al.Hair-bearing human skin generated entirely frompluripotent stem cells.Nature 582,399-404,doi:10.1038/s41586-020-2352-3(2020).
2Lancaster,M.A.&Knoblich,J.A.Generation of cerebral organoids fromhuman pluripotent stem cells.Nature protocols 9,2329-2340,doi:10.1038/nprot.2014.158(2014).
3Leibel,S.L.,McVicar,R.N.,Winquist,A.M.,Niles,W.D.&Snyder,E.Y.Generation of Complete Multi-Cell Type Lung Organoids From HumanEmbryonic and Patient-Specific Induced Pluripotent Stem Cells for InfectiousDisease Modeling and Therapeutics Validation.Curr Protoc Stem Cell Biol 54,e118,doi:10.1002/cpsc.118(2020).
4Sander,V.et al.Protocol for Large-Scale Production of KidneyOrganoids from Human Pluripotent Stem Cells.STAR Protoc 1,100150,doi:10.1016/j.xpro.2020.100150(2020).
5Hofbauer,P.et al.Cardioids reveal self-organizing principles ofhuman cardiogenesis.Cell184,3299-3317e3222,doi:10.1016/j.cell.2021.04.034(2021).
Claims (7)
1.一种人类器官来源外泌体的制备方法，包括下述步骤：
1)以人的诱导多能干细胞或组织来源的成体干细胞或成体分化细胞为起始细胞制备类器官；
2)收集类器官的培养基上清和/或组织解离后的类器官细胞悬液上清，通过超速离心从类器官培养基上清或组织解离后的类器官细胞悬液的上清中分离外泌体。
2.根据权利要求1所述的制备方法，其特征在于：所述类器官是指利用多能干细胞或成体干细胞或成体分化细胞，在体外，通过三维培养方式形成的具有来源器官关键结构和功能的组织类似物。
3.根据权利要求1或2所述的制备方法，其特征在于：所述步骤1)中，所述类器官包括皮肤、脑、肺、肾、心脏或肝脏。
4.根据权利要求1或2所述的制备方法，其特征在于：所述步骤2)中，所述组织解离后的类器官细胞悬液上清的具体制备方法如下：杜氏磷酸盐缓冲液清洗类器官，使用TrypLETMExpress酶于37℃培养箱中消化类器官10-30min，收集细胞悬液于1000-2000r.p.m转速离心5-10min去除细胞，转速3000-5000g离心10-20min去除死细胞，取上清。
5.根据权利要求1或2所述的制备方法，其特征在于：所述步骤2)中，所述超速离心的具体方法为：先低速离心，取上清后超高速离心，弃上清，用1×PBS重悬沉淀后使用0.22μm/0.45μm滤膜过滤，以弃掉上清中的杂质，再次超高速离心；
其中，所述低速离心的条件为：2000-5000g，离心10min，取上清；再10000g，离心30min，离心的温度为4℃；
所述超高速离心的条件为：离心转速为100000g-120000g，离心时间为60-90min，离心的温度为4℃。
6.权利要求1-5中任一项所述方法制备得到的外泌体。
7.权利要求6所述外泌体在制备用于损伤组织修复的产品中的应用。
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