CN116655774A - 过表达tlx的人诱导多能干细胞及其应用 - Google Patents
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Abstract
本发明涉及干细胞培养技术领域，尤其涉及过表达TLX的人诱导多能干细胞及其应用。本发明通过使人诱导多能干细胞(hiPSC)过表达TLX或其截短体，实现hiPSC向NSC的自驱动分化。该方案不仅加快hiPSC向NSC分化，还实现NSC长期稳定体外传代培养，从而为治疗神经***疾病、细胞治疗等提供供体细胞，并且获得的NSC外泌体具有良好的生物学活性。
Description
技术领域
本发明涉及干细胞培养技术领域，尤其涉及过表达TLX的人诱导多能干细胞及其应用。
背景技术
神经干细胞(NSC)是一类能够无限的自我更新并具备分化成神经元，星形胶质细胞和少突胶质细胞的多能细胞，为神经发生发育的研究，神经***疾病模型的建立，药物筛选的***的设计提供了新方法。体外获得NSC的最适合最方便的方法是利用多能干细胞诱导分化。
常用的两种多能干细胞是人诱导多能干细胞(hiPSC)和胚胎干细胞(ESC)。ESC来源于人胚胎细胞，这种细胞的获取由于伦理原因而变得困难。hiPSC通过体细胞重编程表达OCT3/4，SOX2，KLF4和c-Myc而获得，它具有和胚胎干细胞相似的特征，使得hiPSC成为一个可替代ESC的细胞来源。虽然，hiPSC分化神经干性(iNSC)的方法已被广泛研究，但hiPSC获得的iNSC体外培养不稳定，细胞增殖能力丢失以及容易分化，阻碍了iNSC在研究和临床中的应用。因此，hiPSC诱导的iNSC的体外稳定传代培养成为亟需解决的问题。近些年，原代NSC体外长期稳定培养已被广泛研究。常用的永生化原代NSC的方法有体细胞融合，过表达c-Myc，V-Myc，SV40 large T antigen，或者改造端粒酶。这些方法可能会导致NSC丢失表型或者存在致癌性。
近些年以来，对调节干细胞自我更新的细胞内因子(TLX，Sox2，Hes，组蛋白修饰酶，染色体重塑蛋白，小RNA调节器)和细胞外信号分子(如Wnt，Notch，Shh，TGFα，EGF，FGF)已受到广泛关注。其中TLX(也称为NR2E1)是一种孤核受体，在成熟大脑中高表达，特别是SVZ区中的NSC，它对NSC自我更新，增殖和神经发生至关重要。TLX抑制Pten/LSDI，p21等蛋白来维持NSC的自我更新；通过调控SOX2的表达来维持NSC的增殖；此外，TLX可以通过调节Wnt7a，Mmp2，SIRT1等来调控神经发育。Murai等人用表达TLX的慢病毒感染海马体，证明了特异性表达TLX的海马体以自驱动的方式提高NSC的增殖，并促进海马体的神经发生，修复大脑的学习和记忆功能。虽然有报道称TLX过表达与胶质瘤相关，但是Li等人证明了过表达TLX会使神经胶质瘤干细胞的增殖和迁移能力下降，抑制肿瘤发生。
在NSC中过表达TLX，可能获得具备自我更新能力的NSC。现有hiPSC分化NSC的方法的分化周期长，且使用的试剂存在临床安全性问题，获得的NSC细胞阳性率低；并且hiPSC来源的NSC细胞体外无法长期稳定传代，批次间分化获得的NSC重复性低，不能保证足够的高纯度NSC供应用于TLX基因稳定转染表达操作。其他常见的将原代NSC改造为永生化NSC的方法有体细胞融合、过表达c-Myc/V-Myc或SV40 large T antigen，或者改造端粒酶。这些方法可能会导致NSC丢失表型或者存在致癌性。并且，原代NSC本身存在供体伦理争议的问题。因此，目前尚无法获得批次间稳定的规模化培养NSC。这一问题限制了神经***疾病的细胞治疗，更无法获得大量的高质量的NSC源外泌体。
发明内容
有鉴于此，本发明要解决的技术问题在于提供过表达TLX的人诱导多能干细胞及其在分化获得永生化的NSC细胞中应用。
本发明提供了TLX在构建向NSC自驱动分化的hiPSC细胞中的应用；所述TLX包括如下I～IV中的至少一种：
I)、SEQ ID NO:1所示氨基酸序列的TLX蛋白；
II)、在I)所述TLX蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸且与TLX蛋白具有相同或相近功能的蛋白；
III)、编码I)或II)所述蛋白的核酸分子；
IV)、在III)所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
V)、能够调控I)～V)中的至少一种的水平或活性的物质。
本发明还提供了SEQ ID NO:1所示氨基酸序列的TLX蛋白，
或者，本发明提供了TLX蛋白的截短体。本发明所述截短体为SEQ ID NO：1所示氨基酸序列中缺失N端1～200个氨基酸的截短体。
一些实施例中，所述截短体为SEQ ID NO:2所示氨基酸序列的TLX蛋白截短体。
本发明还提供了编码TLX蛋白的核酸。或者本发明提供了编码所述截短体的核酸。
本发明实施例中，编码SEQ ID NO：1所示蛋白的核酸序列如SEQ ID NO:3所示；编码SEQ ID NO：2所示蛋白的核酸序列如SEQ ID NO:4所示。
本发明还提供了一种转录单元，其包括启动子和本发明所述的核酸。
本发明中，所述启动子为CMV启动子或EF1A启动子。
本发明还提供了一种质粒载体，其包括骨架载体和本发明所述的转录单元。
本发明所述的质粒载体中，还可包括编码EGFP的核酸。
EGFP作为报告基因，便于培养效果或分化效果的观测，其表达对细胞生长、TLX的表达及效果发挥不产生影响。
本发明中，对所述骨架载体不做限定，经实验证明，本领域常用于iPSC细胞的质粒载体(例如各种常用的商品化的慢病毒、腺病毒载体，或含有整合酶Integrase的非病毒稳定转染表达载体)都可以用于本发明所述核酸序列的表达。
本发明还提供了一种宿主，其转染或转化有本发明所述的质粒载体。
本发明中所述宿主细胞为大肠杆菌细胞、人肾上皮细胞系、昆虫细胞或hiPSC细胞。
本发明还提供了向NSC自驱动分化的hiPSC细胞，其表达本发明所述的核酸。即该hiPSC细胞中表达编码SEQ ID NO：1所示蛋白的核酸，或者表达编码SEQ ID NO：2所示截短体的核酸。
本发明所述向NSC自驱动分化的hiPSC细胞的构建方法包括：将所述的质粒载体和辅助质粒进行病毒包装，获得慢病毒后感染hiPSC细胞获得向NSC自驱动分化的hiPSC细胞。
本发明还提供了一种永生化NSC细胞，其由本发明所述向NSC自驱动分化的hiPSC细胞分化获得。
本发明还提供了一种hiPSC细胞分化为永生化NSC细胞的方法，其包括，将本发明所述向NSC自驱动分化的hiPSC细胞经培养获得永生化NSC细胞。
本发明中促进hiPSC细胞分化为永生化NSC细胞的培养基，包括基础培养基和BSA、Glutamax添加剂、丙酮酸钠、NaCl、N2营养因子、B27神经营养因子和胰岛素，nonessentialamino acid，FGF2，EGF，heparin中的至少一种。本发明实施例中，所述基础培养基为Neurobasal培养基。
一些实施例中，促进hiPSC细胞分化为永生化NSC细胞的培养基由Neurobasal培养基和1g/100mL～3g/100mL BSA、0.5vol％～1.5vol％Glutamax添加剂、0.5vol％～1.5vol％丙酮酸钠、0.01～0.1mol/L NaCl、2vol％～8vol％N2营养因子、8vol％～12vol％B27神经营养因子、1×青霉素链霉素、8～12ng/mL FGF2和8～12ng/mL胰岛素组成。
一些优选实施例中，促进hiPSC细胞分化为永生化NSC细胞的培养基由Neurobasal培养基和2g/100mL BSA、1vol％Glutamax添加剂、1vol％丙酮酸钠、0.05mol/L NaCl、5vol％N2营养因子、10vol％B27神经营养因子、1×青霉素链霉素培养基、10ng/mL FGF2和10ng/mL胰岛素组成。
本发明还提供了永生化NSC细胞的传代培养方法，所述传代培养的培养基为含有GSK3和TGF-beta抑制剂的培养基。本发明实施例中，所述含有GSK3和TGF-beta抑制剂的培养基为TFM培养基。
本发明还提供了NSC细胞外泌体的制备方法，其包括培养本发明所述的永生化NSC细胞，收集上清液提取获得NSC细胞外泌体。
所述提取方法包括以切向流超滤对所述永生化NSC细胞培养上清进行初纯，再利用Capto Core 700柱(简称Core700)纯化对初纯样品进行精纯。
本发明还提供了所述制备方法制得的NSC细胞外泌体。
本发明所述制备方法制得的NSC细胞外泌体在制备保护神经元的药物中的应用。
本发明还提供了一种保护神经元的药物，其原料所述制备方法制得的NSC细胞外泌体。
本发明还提供了一种保护神经元的方法，其为给予本发明所述的药物。
本发明通过使人诱导多能干细胞(hiPSC)过表达TLX或其截短体，实现hiPSC向NSC的自驱动分化，不仅加快hiPSC向NSC分化，还实现NSC长期稳定体外传代培养，至少传代至45代仍表现出良好干性。从而为获得NSC外泌体提供供体细胞，并且纯化获得具有生物学活性的NSC源外泌体。
附图说明
图1a示lenti-SFH-EGFP-CMV-NR2E1质粒结构；图1b示lenti-SFH-EGFP-CMV-NR2E1(182-386aa)质粒结构；图1c示lenti-CMV-NR2E1质粒结构，图1d示lenti-CMV-NR2E1(182-386aa)质粒结构；图1e示lenti-Ub-EGFP-EF1a-NR2E1质粒结构；图1f示lenti-Ub-EGFP-EF1a-NR2E1(182-386aa)质粒结构；
图2a示蛋白Marker，图2b示Western Blot检测结果；
图3a示hiPSCCMV-FL-TLX细胞形态；图3b示hiPSCEF1A-FL-TLX细胞形态；图3c示两种启动子感染的细胞NR2E1 mRNA表达量；图3d示两种启动子感染的细胞生长曲线；图3e示流式检测两种启动子感染的细胞全长TLX表达量；图3f示流式检测hiPSCTLX-FL的细胞EGFP表达量；
图4a示hiPSCCMV-TP-TLX细胞形态；图4b示hiPSCEF1A-TP-TLX细胞形态；图4c示两种启动子感染的细胞NR2E1 mRNA表达量；图4d示两种启动子感染的细胞生长曲线；图4e示流式检测两种启动子感染的细胞截短体TLX表达量；图4f示流式检测hiPSCTLX-TP的细胞EGFP表达量；
图5示传统NSC分化流程图与本发明分化流程图对比；
图6A不同分化培养基诱导iPSCTLX-FL向iNSCTLX-FL分化，每个培养基对应3幅图，从左至右依次是明场，绿色荧光图，和叠加图，其中，NSC特征形态为花团簇生长；
图6B示不同分化培养基诱导iPSCTLX-TP向iNSCTLX-TP分化，每个培养基对应3幅图，从左至右依次是明场，绿色荧光图，和叠加图，其中，NSC特征形态为花团簇生长；
图6C示NGDI培养基诱导不表达EGFP的iPSCTLX-FL/TP向iNSCTLX-FL/TP分化，其中，NSC特征形态为花团簇生长；
图7示iPSCTLX-FL/TP在NGDI培养基中培养3天，流式细胞术检测NSC蛋白标志物Sox2，Nestin，Vimentin，Musashi的表达；
图8示野生型hiPSC在传统分化方法下分化NSC 21天流式检测结果和过表达TLX的hiPSC在NGDI培养基条件下培养6天NSC流式检测结果；
图9示iNSCTLX细胞在不同维持培养基中培养细胞形态；
图10示iNSCTLX-FL/TP细胞倍增曲线；
图11示Western Blotting检测NSC细胞中Flag标签。NSCFL-lenti，NSCTP-lenti为转染lenti-Ub-EGFP-EF1a-NR2E1或lenti-Ub-EGFP-EF1a-NR2E1(182-386aa)病毒细胞，NSCTP -lenti-EF1a为NSCTP-lenti细胞再转染lenti-Ub-EGFP-EF1a-NR2E1(182-386aa)病毒细胞；
图12示野生型hiPSC分化的NSC细胞P1代和P2代细胞形态照片以及流式检测细胞SOX2/Nestin表达量，iNSCTLX-TP分化的NSC细胞P1代和P15代细胞形态照片以及流式检测细胞SOX2/Nestin表达量；
图13示细胞流式检测iNSCTLX-FL/TP细胞不同代次，SOX2，Nestin，Vimentin，PAX6的表达量；
图14示免疫荧光检测第15代NSCTP-TLX细胞Vimentin，Tuj1，Pax6，Nestin的表达；
图15示左上角是运动神经元细胞形态图，右上角是多巴胺神经元细胞形态图，中间是运动神经元前体细胞免疫荧光结果，最下边是多巴胺神经元前体细胞免疫荧光结果；
图16示运动神经元培养20天(A-C)和多巴胺神经元培养22天(D-F)电生理；其中，A为运动神经元Na+通道电位，B为运动神经元K+通道电位，C为运动神经元诱发动作电位；D为多巴胺神经元Na+通道电位，E为多巴胺神经元Ka+通道电位，F为多巴胺神经元诱发动作电位；
图17示NSC-EV电镜图；
图18示利用纳米流式检测NSC-EV的纯度；
图19示利用纳米流式检测NSC-EV的粒径；
图20示NSC-EV蛋白组学检测到的NSC-EV蛋白，通过与Vesiclepedia数据库进行对比；
图21示通过对NSC-EV的蛋白组学结果进行GO功能分析；
图22示NSC细胞上清经纯化后的形态，其中，A示电镜表征切向流超滤后形态，B示密度梯度离心后组分1#-6#和组分7#-11#的形态，C示QA柱纯化后的形态；
图23示纳米流式检测各个纯化步骤样品：其中，切向流超滤(A)，密度梯度离心组分1#-13#(B)样品，7#组分(C)，QA柱纯化(D)的浓度和纯度；
图24示WB表征精纯后的样品EV标志物CD63和CD81蛋白表达；
图25示1×109p/mL NSC-EV能明显抑制LPS引起的胶质细胞炎症反应；
图26示1×109p/mL NSC-EV能明显抑制神经凋亡。
具体实施方式
本发明提供了过表达TLX的人诱导多能干细胞及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in MolecularBiology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
TLX蛋白全长由385个氨基酸残基组成，序列如SEQ ID NO:1所示，具体为：MSKPAGSTSRILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSGNQGGCPVDKTHRNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIRKQVALYFRGHKEENGAAAHFPSAALPAPAFFTAVTQLEPHGLELAAVSTTPERQTLVSLAQPTPKYPHEVNGTPMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMYKSSDI。
编码TLX蛋白的核酸序列经密码子优化，共计1155bp，序列如SEQ ID NO:3所示，具体为：atgagcaagccagccggatcaacaagccgcattttagatatcccctgcaaagtgtgtggcgaccgcagctcggggaagcactacggggtctacgcctgcgacggctgctcaggttttttcaaacggagcatccgaaggaataggacctatgtctgcaaatctggaaaccagggaggctgtccggtggacaagacgcacagaaaccagtgcagggcgtgtcggctgaagaagtgtttggaagtcaacatgaacaaagacgccgtgcagcacgagcgggggcctcggacgtccaccatccgcaagcaagtggccctctacttccgtggacacaaggaggagaacggggccgccgcgcactttccctcggcggcgctccctgcgccggccttcttcaccgcggtcacgcagctggagccgcacggcctggagctggccgcggtgtccaccactccagagcggcagaccctcgtgagcctggctcagcccacgcccaagtacccccatgaagtgaatgggaccccaatgtatctctatgaagtggccacggagtcggtgtgtgaatcagctgccagacttctcttcatgagcatcaagtgggctaagagtgtgccagccttctccacgctgtctttgcaagaccagctgatgcttttggaagatgcttggagagaactgtttgttctaggaatagcacaatgggccattccggttgatgctaacactctactggctgtatctggcatgaacggtgacaacacagattcccagaagctgaacaagatcatatctgaaatacaggctttacaagaggtggtggctcgatttagacaactccggttagatgctactgaatttgcctgtctaaaatgcatcgtcactttcaaagccgttcctacacatagtggttctgaactgagaagtttccggaatgctgccgccattgcagcccttcaagatgaggctcagctaacgctcaacagctacatccataccagatatcccactcaaccctgtcgctttggaaaactcctgttgcttttgccagctttacgttctattagcccatcaactatagaagaagtgtttttcaaaaaaaccatcggcaatgtgccaattacaagactgctttcagatatgtacaaatccagtgatatc。
本发明研究表明，相对于全长序列而言，TLX的截短体在iPSC细胞中的表达量更高，且表达截短体的hiPSC细胞的增殖速率更高。但相对于野生型hiPSC，不论表达全长序列还是表达截短体序列，经基因改造的hiPSC细胞的增殖速度都得到显著提高。
本发明中，所述截短体相对于TLX氨基酸序列中缺失N端1～200个氨基酸，例如，缺失1～10个、10～20个、20～50个、50～70个、70～100个、100～150个、150～160个、160～170个、170～180个、180～190个或190～200个。
一些实施例中，所述截短体相对于TLX氨基酸序列中缺失N端181个氨基酸，其氨基酸序列如SEQ ID NO:2所示，共有204个氨基酸残基组成，具体为TESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMYKSSDI。
编码SEQ ID NO:2所示TLX蛋白截短体的核酸序列经密码子优化，共计615bp，序列如SEQ ID NO:4所示，具体为atgacggagtcggtgtgtgaatcagctgccagacttctcttcatgagcatcaagtgggctaagagtgtgccagccttctccacgctgtctttgcaagaccagctgatgcttttggaagatgcttggagagaactgtttgttctaggaatagcacaatgggccattccggttgatgctaacactctactggctgtatctggcatgaacggtgacaacacagattcccagaagctgaacaagatcatatctgaaatacaggctttacaagaggtggtggctcgatttagacaactccggttagatgctactgaatttgcctgtctaaaatgcatcgtcactttcaaagccgttcctacacatagtggttctgaactgagaagtttccggaatgctgccgccattgcagcccttcaagatgaggctcagctaacgctcaacagctacatccataccagatatcccactcaaccctgtcgctttggaaaactcctgttgcttttgccagctttacgttctattagcccatcaactatagaagaagtgtttttcaaaaaaaccatcggcaatgtgccaattacaagactgctttcagatatgtacaaatccagtgatatc。
本发明通过实验表明，启动子的选择对于TLX在细胞中的表达量存在显著影响。其中，CMV启动子转染后的iPSC细胞，TLX的表达量明显高于EF1A启动子转染后的细胞的TLX的表达量。
前期研究证明，针对NSC过表达TLX，不仅NSC细胞难以获取，且存在伦理问题。更重要的是，过表达TLX的NSC细胞传代不稳定。而本发明在hiPSC细胞阶段过表达TLX从而获得了稳定表达的hiPSC细胞系，为NSCTLX的获得提供足够数量的细胞种群；并且在实验中发现，TLX可能对hiPSC向NSC的分化具有促进作用。因此，在本发明的方案中，对于hiPSCTLX而言，向NSCTLX转化仅需要六天的时间。但对于不过表达TLX的野生型hiPSC细胞在相同的培养基无法实现向NSC细胞的转化。或者，将野生型hiPSC细胞在NGD培养基中额外再加入SMAD小分子抑制剂(compound C)，培养9天，才能够获得低表达Nestin,Sox2的细胞，获得高纯度的野生型hiPSC分化的NSC细胞种群通常需要14-21天。
本发明提供的向NSC自驱动分化的hiPSC细胞中，表达本发明所述的核酸。即该hiPSC细胞中表达编码SEQ ID NO：1所示蛋白的核酸，或者表达编码SEQ ID NO：2所示截短体的核酸。
本发明中，利用慢病毒感染hiPSC，获得了可稳定传代的hiPSCTLX，并证明了TLX可作为hiPSC向NSC分化的内驱动力，驱动hiPSC向NSC分化，获得可长期维持自我更新的NSCTLX。本发明中，过表达TLX的hiPSC在NGDI培养条件下，获得的NSC的生物标志物的表达量远高于野生型hiPSC在传统分化条件下获得的NSC生物标志物的表达量
本发明中，培养hiPSCTLX细胞转染截短体TLX病毒的hiPSC分化的NSC细胞，能维持稳定传代，并且能够维持良好的干性和分化潜能。
本发明中，通过体外稳定维持NSC细胞传代，收集NSC细胞上清，利用现有外泌体提取技术，提取NSC细胞上清外泌体，经检测该外泌体中含有和免疫调节，信号转导，细胞间通讯等生物学过程相关的蛋白，并且还含有大量和神经***相关的miRNA。
经过试验验证，本发明所述的NSC细胞的外泌体对神经元具有良好的保护效果。经LPS刺激后的胶质细胞的培养上清对神经细胞有杀伤作用，但将NSC-EV加入该培养伤情后，神经元凋亡明显低于不含NSC-EV的对照组。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1 TLX(或记做NR2E1)质粒构建及病毒包装
TLX能够维持NSC的增殖和干性。利用病毒能够基因重组的原理，构建携带TLX基因的慢病毒质粒，并将质粒转入慢病毒中。
慢病毒载体是以HIV-1为基础发展起来的基因治疗载体。区别于一般的逆转录病毒载体，它对***细胞和非分***细胞均具有感染能力。
慢病毒载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。在感染能力方面可有效地感染神经元细胞，干细胞等多种类型的细胞。对于一些以其他手段较难转染的细胞，如原代细胞，干细胞等，使用慢病毒载体，能大大提高目的基因的转导效率，且目的基因整合到宿主细胞基因组中的几率大大增加，能较方便快捷地实现目的基因的长期，稳定表达。
1、实验步骤：
1)获取目的基因片段：使用高保真的PrimeSTAR酶扩增目的基因(核酸SEQ ID NO:3～4所示。其中SEQ ID NO:3编码SEQ ID NO:1所示TLX全长，记为TLX-FL。SEQ ID NO:4编码SEQ ID NO:2的TLX截短体，记为TLX-TP。)，PCR产物进行琼脂糖凝胶电泳检测扩增效果，并把目的基因条带从琼脂糖凝胶电泳后的胶中割下来，用胶回收试剂盒进行胶回收。另将目的基因与编码绿色荧光蛋白(EGFP)的基因连接，获得融合片段。
2)线性化表达载体的制备：用限制性内切酶对表达载体进行酶切，酶切反应体系为：2μg质粒，10×反应buffer 5μL，限制性内切酶各1μL，用水补足50μL，于37℃水浴锅中孵育2h以上。酶切产物进行琼脂糖凝胶电泳检测酶切效果，并把目的载体条带从琼脂糖凝胶电泳后的胶中割下来，用胶回收试剂盒进行胶回收。
3)目的基因构建入线性化表达载体中：使用无缝组装试剂盒，将目的基因片段和线性化载体以摩尔比1:1加入到离心管中，混合后在37℃孵育30min，然后转移到冰上5min。
4)感受态细胞转化，将构建好的表达载体转化入大肠杆菌感受态细胞。
5)菌落PCR鉴定阳性转化子：挑取平板上长出的转化子重悬于10μL LB培养液中，取1μL作为模板进行菌落PCR鉴定。
6)阳性克隆进行测序。
7)使用质粒小提试剂盒提取质粒。
8)病毒包装：将4×106细胞接种100mm皿中，密度为70％-80％。一天后，将病毒载体质粒和转染试剂加入到细胞培养板中，6h后，更换新鲜的完全培养基。分别48h和72h收病毒，进行纯化。
9)病毒验证：将获得的病毒感染293T细胞，用WB验证目的基因的表达情况。
实验结果如图1～2：
实验结论：前述方法质粒构建成功(质粒图谱如图1a～图1f所示)，并且病毒能够实现细胞转染，使细胞表达目的基因(图2a和图2b)。
实施例2 TLX慢病毒感染hiPSC细胞
利用慢病毒感染hiPSC，使hiPSC过表达TLX，获得稳定传代的hiPSCTLX细胞株，实验步骤包括：
1)细胞按2.5×105/well接种12孔板，培养24h。
2)将细胞培养基更换为含有病毒的新鲜培养基，病毒计算按MOI＝10进行计算。其中，lenti-SFH-EGFP-CMV-NR2E1转染获得的细胞记为hiPSCFL-TLX；lenti-SFH-EGFP-CMV-NR2E1(182-386aa)转染获得的细胞记为hiPSCTP-TLX。
3)细胞培养16h后，移除旧培养基，将培养基更换为新鲜培养基，每天换液，每隔3天，细胞传代一次。
实验结果与结论：
图3a和图3b表明，细胞成功感染TLX病毒，同时证明过表达全长TLX不影响iPSC的形态，图3c通过qPCR检测到感染后的hiPSC高表达TLX全长基因，且两种启动子中，CMV启动子更有利于全长TLX的表达。图3d表明，不论以哪种启动子进行过表达，全长TLX的表达均不影响iPSC的生长。且感染不同启动子的病毒，两种启动子均能使全长TLX在hiPSC细胞中表达(图3e)。且EGFP的表达对细胞生长和TLX功能的发挥无影响(图3f)。
图4a和图4b表明，细胞成功感染TLX病毒，同时证明过表达截短体TLX不影响iPSC的形态。图4c通过qPCR检测到感染后的hiPSC高表达TLX截短体基因，且两种启动子中，CMV启动子更有利于TLX截短体的表达。图4d表明，不论以哪种启动子进行过表达，TLX截短体的表达均不影响iPSC的生长。且感染不同启动子的病毒，两种启动子均能使截短体TLX在hiPSC细胞中表达(图4e)。另根据图4f，EGFP的表达对细胞生长和TLX功能的发挥无影响。
实施例3 hiPSCTLX向NSC自驱动分化
实验目的：利用iPSC内外源过表达的TLX或TLX截短体驱动iPSC在NSC培养基条件下，实现iPSC向NSC的分化，以未经转染的野生型iPSC为对照。采用的iPSC细胞表达EGFP，并过表达TLX或TLX截短体。其中，EGFP荧光蛋白做为报告基因用于观察培养基的培养效果，另对表达TLX或TLX截短体但不表达EGFP的细胞进行培养，证明EGFP的表达对培养效果无影响。
实验步骤：
按照细胞和培养基的不同将实验分为如下几组：
对照：不过表达TLX的hiPSC的分化：第0～7天以SB431542，LDN193189和LIF诱导分化至第7日，更换SB431542继续诱导至第14天，以FGF2和EGF诱导至第21日。
组a(NGD)：培养基由Neurobasal培养基和2g/100mL BSA、1vol％Glutamax添加剂、1vol％丙酮酸钠、0.05mol/L NaCl、5vol％N2营养因子、10vol％B27神经营养因子和1×青霉素链霉素。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现NSC的典型形态——Rosette(花团簇生长)样；
组b(NGD-F)：培养基NGD中加入10ng/mL FGF。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞中出现NSC Rosette(花团簇生长)样；
组c(NGD-C)：培养基NGD中加入2.5μM Compound C。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组d(NGD-L)：培养基NGD中加入10ng/mL LIF。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组e(NGD-FI)：培养基NGD中加入10ng/mL FGF2和10ng/mL胰岛素。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组f(NGD-I)：培养基NGD中加入10ng/mL FGF2和10ng/mL胰岛素。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组g(NGD-FIL)：培养基NGD中加入10ng/mL FGF2，10ng/mL胰岛素和10ng/mL LIF。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组h(LSL)：ncEpic培养基中加入10μM SB431542，0.25μM LDN193189和10ng/mLLIF；细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞死亡；
组i(LSDL)：ncEpic培养基中加入10μM SB431542，0.25μM LDN193189，2.5μM DMH-1和10ng/mL LIF；细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞死亡；
组j(NIM)：DMEM/F12培养基中加入0.5mL N2，0.5mL Glutamax，0.5mL NEAA和10ng/mL Heparin。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
组k(NIM++)：DMEM/F12培养基中加入0.5mL N2，0.5mL Glutamax，0.5mL NEAA，10ng/mL Heparin，10ng/mL FGF2和10ng/mL EGF。细胞接种到6孔板，2.5×105cells/well，接种时培养基为Epic培养基，第二天，将培养基更换为组a培养基，培养3天后，细胞未出现Rosette(花团簇生长)样；
实验结果与结论(图5～6)：
如图5分化流程图，以NGDI培养基为例，本发明与传统hiPSC分化NSC方法相比，明显缩短分化时间，并明显简化了分化步骤和所用的试剂。
不同分化培养基诱导iPSCTLX-FL向iNSCTLX-FL分化，只有NGD-F和NGD-I，NGD-L培养基培养的细胞出现rosette形态。经过多次重复，NGD-I培养基的重复效果最好。传统分化NSC培养基(SLS和LSDL)培养的细胞，能明显看到细胞大量死亡，培养3天后，几乎所有细胞脱落死亡(图6A)。
不同分化培养基诱导iPSCTLX-TP向iNSCTLX-TP分化，只有NGD-F和NGD-I培养基培养的细胞出现rosette形态。经过多次重复，NGD-I培养基的重复效果最好。传统分化NSC培养基(SLS和LSDL)培养的细胞，能明显看到细胞大量死亡，培养3天后，几乎所有细胞脱落死亡(图6B)。
此外，不含EGFP基因的过表达TLX的hiPSC在NGD-I培养基中培养3天后，出现了NSCrosette形态特征(图6C)，利用流式对该状态细胞进行检测，细胞表达NSC标志蛋白(Nestin，Sox2，Vimentin，Musashi)(图7)。
如图8，过表达TLX的hiPSC在NGDI培养基中培养6天，Nestin+/Sox2+，Vimentin+/Musashi+双阳性细胞比例明显野生型hiPSC利用传统分化方法分化21天的细胞比例，说明本发明方法能在很短的实验周期获得比传统方法更高阳性率的NSC细胞。
实施例4 NSC稳定传代培养
为了实现NSCTLX长期稳定传代培养，将实施例3分化获得的NSCTLX-FL、NSCTLX-TP细胞或对照组分化获得的细胞分别按0.25×105cells/cm2传代，每5天传代一次(根据选择的培养基分组)。用于传代培养的细胞不表达EGFP，但表达TLX或TLX截短体。孔板包被材料为Matrigel，Vitronectin或Laminin。
组1(NGD-I)：培养基由Neurobasal培养基和2g/100mL BSA、1vol％Glutamax添加剂、1vol％丙酮酸钠、0.05mol/LNaCl、5vol％N2营养因子、10vol％B27神经营养因子和1×青霉素链霉素，加入10ng/mL胰岛素。
组2(NIM)：DMEM/F12培养基中加入0.5mL N2，0.5mL Glutamax，0.5mL NEAA和10ng/mL Heparin。
组3(NIM++)：DMEM/F12培养基中加入0.5mL N2，0.5mL Glutamax，0.5mL NEAA和10ng/mL Heparin，10ng/mL FGF2和10ng/mL EGF，细胞能稳定传代。
组4(TFM)：DMEM/F12培养基和Neurobasal培养基比例1:1混合后，加入2％TFM添加剂(含GSK3和TGF-β抑制剂)。细胞能长期稳定传代。
结果表明(图9)，iNSCTLX在4种维持培养基中，均能较好的维持传代，其中，当培养基中含有GSK3和TGF-beta通路抑制剂时(本案例中，以能购买到的含有GSK3和TGF-β抑制剂的商品化培养基为例进行说明，即TFM培养基)，能更好的维持iNSCTLX细胞传代稳定性。
图10所示，监测iNSCTLX的生长速率，iNSCTLX均能稳定高速增殖传代，此外，iNSCTLX -TP的生长速率明显高于iNSCTLX-FL。
图11所示，WesternBlotting检测NSC细胞中Flag标签。NSCFL-lenti，NSCTP-lenti为转染lenti-Ub-EGFP-EF1a-NR2E1或lenti-Ub-EGFP-EF1a-NR2E1(182-386aa)病毒细胞，NSCTP -lenti-EF1a为NSCTP-lenti细胞再转染lenti-Ub-EGFP-EF1a-NR2E1(182-386aa)病毒细胞，Flag标签与NR2E1融合表达，通过检测Flag标签的表达，说明hiPSC中转染的TLX，在NSC细胞中仍能稳定表达。
图12所示，野生型hiPSC分化的NSC细胞P1代和P2代细胞形态照片以及流式检测细胞Sox2/Nestin表达量，以及iNSCTLX-TP细胞P1代和P15代细胞形态照片以及流式检测细胞Sox2/Nestin表达量，野生型iNSC细胞传至P2细胞明显出现神经元分化，NSC蛋白标志物表达下降，同时结合图13所示，流式检测不同代次iNSCTLX细胞中SOX2，Nestin，Vimentin，PAX6的表达量，iNSCTLX-TP细胞传至45代，SOX2，Nestin，Vimentin，PAX6蛋白仍能稳定表达。说明本发明实现了NSC传代从1到至少45代传代的突破。
此外，利用免疫荧光检测，iNSCTLX-TP细胞Vimentin，Tuj1，Pax6，Nestin的表达，再次证明iNSCTLX-TP高表达NSC生物标志物(Vimentin，Pax6，Nestin)，不表达神经元标志物(Tuj1)(图14)。
实施例5 NSC干性维持表征
为了证明过表达TLX不会影响NSC细胞的干性，利用多巴胺神经元和运动神经分化试剂盒，分别将NSC细胞分化为多巴胺神经元和运动神经元
图15左上角是运动神经元细胞形态图，右上角是多巴胺神经元细胞形态图。中间是运动神经元前体细胞免疫荧光结果。最下边是多巴胺神经元前体细胞免疫荧光结果。从免疫荧光结果上显示，NSC能成功分化为多巴胺和运动神经元前体细胞。过表达TLX不影响NSC细胞的干性。
图16运动神经元培养20天(A-C)和多巴胺神经元培养22天(D-F)电生理。其中，其中，A为运动神经元Na+通道电位，B为运动神经元K+通道电位，C为运动神经元诱发动作电位；D为多巴胺神经元Na+通道电位，E为多巴胺神经元K+通道电位，F为多巴胺神经元诱发动作电位。说明运动神经元和多巴胺神经元已分化成熟。
实施例6 NSC源外泌体提取及表征
通过体外稳定维持NSC细胞传代，收集NSC细胞上清，利用现有外泌体提取技术，提取NSC细胞上清外泌体
实验方法：利用切向流超滤对NSC细胞上清进行初纯，再利用Core700柱纯化对初纯样品进行精纯
实验结果与结论：图17示NSC-EV电镜图，结果显示培养获得的NSC-EV具有良好的形态。利用纳米流式检测NSC-EV的纯度高达87.87％(图18)利用纳米流式检测NSC-EV的粒径，粒径分布为62.01±14.21nm(图19)。NSC-EV蛋白组学检测到的NSC-EV蛋白，通过与Vesiclepedia数据库进行对比，NSC-EV蛋白组学中检测到的高达93.66％的蛋白属于细胞外囊泡，说明纯化方法的可靠性(图20)。通过对NSC-EV的蛋白组学结果进行GO分析，分别分析了生物学过程(biological process)(图21)
结论：从结果上看，NSC-EV的蛋白中含有和免疫调节，信号转导，细胞交流等生物学过程相关的蛋白。
表1通过对NSC-EV转录组学检测，NSC-EV中含有大量和神经***相关的miRNA。
实施例7 NSC-EV纯化及表征
通过体外稳定维持NSC细胞传代，收集NSC细胞上清，利用现有外泌体提取技术，提取NSC细胞上清外泌体
实验方法：利用切向流超滤对NSC细胞上清进行初纯，再利用密度梯度离心对样品进一步纯化，最后利用QA柱对初纯样品进行精纯
实验结果：
图22为电镜表征切向流超滤(A)，密度梯度离心组分1#-6#和组分7#-11#(B)，QA柱纯化(C)的形态，说明随着纯化，样品中的杂蛋白逐步除去。
图23纳米流式检测各个纯化步骤样品：切向流超滤(A)，7#组分(C)，QA柱纯化(D)的浓度和纯度，以及密度梯度离心组分1#-13#(B)样品浓度。随着纯化，样品的纯度逐步提高。
图24WB表征精纯后的样品EV标志物CD63和CD81蛋白表达，以及以NSC细胞作为对照，表征NSC-EV中Flag的表达。说明得到的样品为高纯度的EV，并且不携带过表达的TLX蛋白。
实施例8 NSC-EV药效评价
通过NSC-EV组学分析结果，NSC-EV中含有和神经***相关的miRNA以及和免疫相关的蛋白，通过构建的细胞外神经***炎症模型，验证NSC-EV对神经元的保护效果
实验方法：利用LPS和不同浓度的NSC-EV共培养大鼠原代胶质细胞24h后，胶质细胞收集用于qPCR检测炎症因子基因的变化；通过共培养小室，将大鼠原代胶质细胞(小室中培养)与大鼠原代神经元细胞(6孔板中)共培养，72h后，细胞流式检测神经元细胞的凋亡情况
实验结果如图25，结果表明，加入1×109p/mL NSC-EV组的细胞的炎症基因IL-1α，IL-1β和TNF-α的表达量与对照组LPS组相比，表达量明显下降，说明1×109p/mL NSC-EV能明显抑制LPS引起的胶质细胞炎症反应。
实验结果如图26，结果表明：1×109p/mL NSC-EV与LPS共培养胶质细胞的上清培养的神经元的凋亡细胞明显低于LPS组。说明NSC-EV对神经元具有神经保护效果。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京桑吉梅拉科技中心（有限合伙）
<120> 过表达TLX的人诱导多能干细胞及其应用
<130> MP21030964
<160> 4
<170> SIPOSequenceListing 1.0
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Thr Tyr Val Cys Lys Ser Gly Asn Gln Gly Gly Cys Pro Val Asp Lys
50 55 60
Thr His Arg Asn Gln Cys Arg Ala Cys Arg Leu Lys Lys Cys Leu Glu
65 70 75 80
Val Asn Met Asn Lys Asp Ala Val Gln His Glu Arg Gly Pro Arg Thr
85 90 95
Ser Thr Ile Arg Lys Gln Val Ala Leu Tyr Phe Arg Gly His Lys Glu
100 105 110
Glu Asn Gly Ala Ala Ala His Phe Pro Ser Ala Ala Leu Pro Ala Pro
115 120 125
Ala Phe Phe Thr Ala Val Thr Gln Leu Glu Pro His Gly Leu Glu Leu
130 135 140
Ala Ala Val Ser Thr Thr Pro Glu Arg Gln Thr Leu Val Ser Leu Ala
145 150 155 160
Gln Pro Thr Pro Lys Tyr Pro His Glu Val Asn Gly Thr Pro Met Tyr
165 170 175
Leu Tyr Glu Val Ala Thr Glu Ser Val Cys Glu Ser Ala Ala Arg Leu
180 185 190
Leu Phe Met Ser Ile Lys Trp Ala Lys Ser Val Pro Ala Phe Ser Thr
195 200 205
Leu Ser Leu Gln Asp Gln Leu Met Leu Leu Glu Asp Ala Trp Arg Glu
210 215 220
Leu Phe Val Leu Gly Ile Ala Gln Trp Ala Ile Pro Val Asp Ala Asn
225 230 235 240
Thr Leu Leu Ala Val Ser Gly Met Asn Gly Asp Asn Thr Asp Ser Gln
245 250 255
Lys Leu Asn Lys Ile Ile Ser Glu Ile Gln Ala Leu Gln Glu Val Val
260 265 270
Ala Arg Phe Arg Gln Leu Arg Leu Asp Ala Thr Glu Phe Ala Cys Leu
275 280 285
Lys Cys Ile Val Thr Phe Lys Ala Val Pro Thr His Ser Gly Ser Glu
290 295 300
Leu Arg Ser Phe Arg Asn Ala Ala Ala Ile Ala Ala Leu Gln Asp Glu
305 310 315 320
Ala Gln Leu Thr Leu Asn Ser Tyr Ile His Thr Arg Tyr Pro Thr Gln
325 330 335
Pro Cys Arg Phe Gly Lys Leu Leu Leu Leu Leu Pro Ala Leu Arg Ser
340 345 350
Ile Ser Pro Ser Thr Ile Glu Glu Val Phe Phe Lys Lys Thr Ile Gly
355 360 365
Asn Val Pro Ile Thr Arg Leu Leu Ser Asp Met Tyr Lys Ser Ser Asp
370 375 380
Ile
385
<210> 2
<211> 204
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Thr Glu Ser Val Cys Glu Ser Ala Ala Arg Leu Leu Phe Met Ser Ile
1 5 10 15
Lys Trp Ala Lys Ser Val Pro Ala Phe Ser Thr Leu Ser Leu Gln Asp
20 25 30
Gln Leu Met Leu Leu Glu Asp Ala Trp Arg Glu Leu Phe Val Leu Gly
35 40 45
Ile Ala Gln Trp Ala Ile Pro Val Asp Ala Asn Thr Leu Leu Ala Val
50 55 60
Ser Gly Met Asn Gly Asp Asn Thr Asp Ser Gln Lys Leu Asn Lys Ile
65 70 75 80
Ile Ser Glu Ile Gln Ala Leu Gln Glu Val Val Ala Arg Phe Arg Gln
85 90 95
Leu Arg Leu Asp Ala Thr Glu Phe Ala Cys Leu Lys Cys Ile Val Thr
100 105 110
Phe Lys Ala Val Pro Thr His Ser Gly Ser Glu Leu Arg Ser Phe Arg
115 120 125
Asn Ala Ala Ala Ile Ala Ala Leu Gln Asp Glu Ala Gln Leu Thr Leu
130 135 140
Asn Ser Tyr Ile His Thr Arg Tyr Pro Thr Gln Pro Cys Arg Phe Gly
145 150 155 160
Lys Leu Leu Leu Leu Leu Pro Ala Leu Arg Ser Ile Ser Pro Ser Thr
165 170 175
Ile Glu Glu Val Phe Phe Lys Lys Thr Ile Gly Asn Val Pro Ile Thr
180 185 190
Arg Leu Leu Ser Asp Met Tyr Lys Ser Ser Asp Ile
195 200
<210> 3
<211> 1155
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgagcaagc cagccggatc aacaagccgc attttagata tcccctgcaa agtgtgtggc 60
gaccgcagct cggggaagca ctacggggtc tacgcctgcg acggctgctc aggttttttc 120
aaacggagca tccgaaggaa taggacctat gtctgcaaat ctggaaacca gggaggctgt 180
ccggtggaca agacgcacag aaaccagtgc agggcgtgtc ggctgaagaa gtgtttggaa 240
gtcaacatga acaaagacgc cgtgcagcac gagcgggggc ctcggacgtc caccatccgc 300
aagcaagtgg ccctctactt ccgtggacac aaggaggaga acggggccgc cgcgcacttt 360
ccctcggcgg cgctccctgc gccggccttc ttcaccgcgg tcacgcagct ggagccgcac 420
ggcctggagc tggccgcggt gtccaccact ccagagcggc agaccctcgt gagcctggct 480
cagcccacgc ccaagtaccc ccatgaagtg aatgggaccc caatgtatct ctatgaagtg 540
gccacggagt cggtgtgtga atcagctgcc agacttctct tcatgagcat caagtgggct 600
aagagtgtgc cagccttctc cacgctgtct ttgcaagacc agctgatgct tttggaagat 660
gcttggagag aactgtttgt tctaggaata gcacaatggg ccattccggt tgatgctaac 720
actctactgg ctgtatctgg catgaacggt gacaacacag attcccagaa gctgaacaag 780
atcatatctg aaatacaggc tttacaagag gtggtggctc gatttagaca actccggtta 840
gatgctactg aatttgcctg tctaaaatgc atcgtcactt tcaaagccgt tcctacacat 900
agtggttctg aactgagaag tttccggaat gctgccgcca ttgcagccct tcaagatgag 960
gctcagctaa cgctcaacag ctacatccat accagatatc ccactcaacc ctgtcgcttt 1020
ggaaaactcc tgttgctttt gccagcttta cgttctatta gcccatcaac tatagaagaa 1080
gtgtttttca aaaaaaccat cggcaatgtg ccaattacaa gactgctttc agatatgtac 1140
aaatccagtg atatc 1155
<210> 4
<211> 615
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgacggagt cggtgtgtga atcagctgcc agacttctct tcatgagcat caagtgggct 60
aagagtgtgc cagccttctc cacgctgtct ttgcaagacc agctgatgct tttggaagat 120
gcttggagag aactgtttgt tctaggaata gcacaatggg ccattccggt tgatgctaac 180
actctactgg ctgtatctgg catgaacggt gacaacacag attcccagaa gctgaacaag 240
atcatatctg aaatacaggc tttacaagag gtggtggctc gatttagaca actccggtta 300
gatgctactg aatttgcctg tctaaaatgc atcgtcactt tcaaagccgt tcctacacat 360
agtggttctg aactgagaag tttccggaat gctgccgcca ttgcagccct tcaagatgag 420
gctcagctaa cgctcaacag ctacatccat accagatatc ccactcaacc ctgtcgcttt 480
ggaaaactcc tgttgctttt gccagcttta cgttctatta gcccatcaac tatagaagaa 540
gtgtttttca aaaaaaccat cggcaatgtg ccaattacaa gactgctttc agatatgtac 600
aaatccagtg atatc 615
Claims (20)
1.TLX在构建向NSC自驱动分化的hiPSC细胞中的应用；所述TLX包括如下I～IV中的至少一种：
I)、SEQ ID NO:1所示氨基酸序列的TLX蛋白；
II)、在I)所述TLX蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸且与TLX蛋白具有相同或相近功能的蛋白；
III)、编码I)或II)所述蛋白的核酸分子；
IV)、在III)所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
V)、能够调控I)～V)中的至少一种的水平或活性的物质。
2.蛋白，其特征在于，
为SEQ ID NO:1所示氨基酸序列的TLX蛋白，
或为SEQ ID NO：1所示氨基酸序列中缺失N端1～200个氨基酸的截短体。
3.根据权利要求2所述的蛋白，其特征在于，其为SEQ ID NO:2所示氨基酸序列的TLX蛋白截短体。
4.编码权利要求2或3所述蛋白的核酸。
5.根据权利要求4所述的核酸，其特征在于，
编码SEQ ID NO：1所示蛋白的核酸序列如SEQ ID NO:3所示；
编码SEQ ID NO：2所示蛋白的核酸序列如SEQ ID NO:4所示。
6.转录单元，其特征在于，包括启动子和权利要求4或5所述的核酸。
7.根据权利要求6所述的转录单元，其特征在于，所述启动子为CMV启动子或EF1A启动子。
8.质粒载体，其特征在于，包括骨架载体和权利要求6或7所述的转录单元。
9.宿主，其特征在于，转染或转化有权利要求8所述的质粒载体。
10.向NSC自驱动分化的hiPSC细胞，其特征在于，其表达权利要求4或5所述核酸。
11.权利要求10所述hiPSC细胞的构建方法，其特征在于，包括：将权利要求8所述的质粒载体和辅助质粒进行病毒包装，获得慢病毒后感染hiPSC细胞获得向NSC自驱动分化的hiPSC细胞。
12.永生化NSC细胞，其特征在于，其由权利要求10所述的hiPSC细胞分化获得。
13.培养基，其特征在于，包括基础培养基和BSA、Glutamax添加剂、丙酮酸钠、NaCl、N2营养因子、B27神经营养因子、胰岛素、nonessential amino acid、FGF2、EGF或heparin中的一种或两种以上的组合。
14.hiPSC细胞分化为永生化NSC细胞的方法，其特征在于，将权利要求10所述的hiPSC细胞经培养获得永生化NSC细胞。
15.根据权利要求14所述的方法，其特征在于，所述培养的培养基为权利要求13所述的培养基。
16.权利要求12所述的永生化NSC细胞的传代培养方法，其特征在于，所述传代培养的培养基为含有GSK3和TGF-beta抑制剂的培养基。
17.NSC细胞外泌体的制备方法，其特征在于，包括培养权利要求12所述的永生化NSC细胞，收集上清液提取获得NSC细胞外泌体。
18.权利要求17所述制备方法制得的NSC细胞外泌体。
19.权利要求17所述制备方法制得的NSC细胞外泌体在制备保护神经元的药物中的应用。
20.一种保护神经元的药物，其特征在于，其原料包括权利要求17所述制备方法制得的NSC细胞外泌体。
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| PCT/CN2023/076366 WO2023155825A1 (zh) | 2022-02-18 | 2023-02-16 | 过表达tlx的人诱导多能干细胞及其应用 |
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