CN113652402A - 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 - Google Patents
一种诱导胶质细胞转分化为功能性神经元的方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种诱导胶质细胞转分化为功能性神经元的方法及其应用。具体地，提供了一种Neurog2功能性片段的用途，该功能性片段可在体内或体外诱导胶质细胞形成功能性神经元细胞，不仅可以在正常组织起到转分化的作用，还可以促进受损神经组织的神经重建，有望成为一种在体神经组织修复的新方法。
Description
技术领域
本发明属于生物技术和基因治疗领域，具体地，本发明涉及一种诱导胶质细胞转分化为功能性神经元细胞的方法及应用，特别是在脊髓损伤修复中的应用。
背景技术
哺乳动物的中枢神经系统损伤和多种神经退行性疾病引起的主要病理变化是不可逆的神经元变性坏死和神经环路破坏。如何补充替代损伤和疾病的大脑或脊髓中死亡丢失的神经元并重建神经环路是治疗的关键步骤。由于成年哺乳动物的中枢神经系统(大脑和脊髓)自我修复的能力非常有限，很难靠自身来弥补神经元细胞的丢失。
由于外源性神经元或神经源性细胞的移植的效率较低，而且有致瘤性和免疫原性的潜在风险。近年来，细胞重编程技术的出现给再生医学带来了革命性的变化。通过表达单个或组合的多个神经原性转录因子实现体内重编程星形胶质细胞诱导产生神经元有望成为神经元替代疗法的重要新策略。因此，寻找合适的转录因子，在体内诱导星形胶质细胞转分化为有活性的神经元，对于大脑和脊髓功能修复非常地重要。特别是目前，尚无有效逆转脊髓损伤的神经元修复方法。
发明内容
本发明提供了Neurog2功能性片段在诱导体外或体内胶质细胞转分化为功能性神经元细胞的方法，以及其在制备神经修复药物组合物方面的应用。
本发明第一方面，提供了一种Neurog2功能性片段的用途，(i)用于制备神经诱导胶质细胞形成功能性神经元细胞的药物组合物；和/或(ii)用于制备针对神经系统疾病的药物组合物。
其中，所述的胶质细胞选自来源于人或非人哺乳动物的星形胶质细胞、NG2胶质细胞、少突胶质细胞、小胶质细胞等，优选地，所述的胶质细胞为星形胶质细胞。
在另一优选例中，所述的星形胶质细胞包括正常状态和损伤状态下的星形胶质细胞。所述的损伤状态为机械性的创伤、中风、神经退行性疾病或其他神经系统疾病引起的神经元死亡、凋亡从而致使神经信号传导受阻或紊乱。
在另一优选例中，所述的星形胶质细胞来源于脊髓、背侧中脑或大脑皮层，较佳地，所述的星形胶质细胞来源于脊髓和背侧中脑。
所述的功能性神经元为具有神经元形态、具有一种或多种神经元标记物且能够发放动作电位并形成突触联系等特征；所述的功能性神经元还可以是神经元细胞群，具体地，神经元细胞群具有以下一种或多种特征：
(a)至少50％的神经元细胞，优选至少60％、70％、80％、90％或100％的神经元细胞表达成熟神经元的标志物NeuN；
(b)能够发放动作电位并能够形成突触联系。
在另一优选例中，所述的功能性神经元包括谷氨酸能神经元或谷氨酸能神经元群。
在另一优选例中，所述的功能性神经元能够发放动作电位并能够形成突触联系。
所述的Neurog2功能性片段为功能性的Neurog2蛋白或编码Neurog2功能性蛋白的核酸序列；所述的Neurog2功能性片段为可正常执行Neurogenin 2转录因子生理功能的蛋白或核酸序列；优选地，Neurog2来源于哺乳动物，较佳地，来源于人或非人灵长类哺乳动物。
在另一优选例中，所述Neurog2功能性片段记载在GenBank号为11924，蛋白序列如SEQ ID NO.:1所示；编码所述的Neurog2基因的mRNA NCBI Reference Sequence号为NM_009718.3，CDS序列如SEQ ID NO.:2所示。
在另一优选例中，所述Neurog2功能性片段记载在GenBank号为63973，蛋白序列如SEQ ID NO.:3所示；编码所述的Neurog2基因的mRNA NCBI Reference Sequence号为NM_024019.4，CDS序列如SEQ ID NO.:4所示。
在另一优选例中，所述的Neurog2的功能性片段序列与SEQ ID NO.:1的序列同源性不低于83％；更优地，所述的Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于90％；最优地，所述的Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于95％。
在另一优选例中，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于80％；更优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于90％；最优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于95％。
在另一优选例中，所述的Neurog2的功能性片段序列与SEQ ID NO.:3的序列同源性不低于83％；更优地，所述的Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于90％；最优地，所述的Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于95％。
在另一优选例中，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于80％；更优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于90％；最优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于95％。
本发明第二方面，提供了载有Neurog2功能性片段的递送系统。
所述的递送系统可以应用于体外或应用于体内诱导胶质细胞转分化为功能性神经元细胞；所述的胶质细胞来源于脊髓、背侧中脑或大脑皮层，较佳地，所述的胶质细胞来源于脊髓和背侧中脑；所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
所述的Neurog2功能性片段可以依靠胶质细胞被动吸收或通过递送系统到达胶质细胞内部起效，所述的递送系统载有Neurog2功能性片段，包括但不限于载有Neurog2功能性片段的表达载体、包裹有Neurog2功能性片段的纳米颗粒、包裹有Neurog2功能性片段的外泌体、包裹有Neurog2功能性片段的改造红细胞或细菌、挂载有Neurog2功能性片段的靶向效应物(如胶质细胞特异性抗体、多肽或其他靶向物质等)。
在一优选例中，所述的递送系统为载有Neurog2功能性片段的表达载体，所述的表达载体可进入胶质细胞，并在星形胶质细胞中表达外源的Neurog2蛋白。
在另一优选例中，所述的表达载体包括质粒、病毒载体；优选为腺相关病毒载体(AAV)，包括但不限于腺病毒载体、逆转录病毒表达载体或慢病毒载体等；
在一优选例中，所述的载有Neurog2功能性片段的表达载体中还载有胶质细胞特异性的启动子，所述的启动子，包括但不限于GFAP启动子、NG2启动子、Aldh1L1启动子、IBA1启动子、CNP启动子、LCN2启动子或经过基因工程改造后的启动子变体。
在一优选例中，所述的载有Neurog2功能性片段的表达载体中还载有一个或多个调控基因表达的调控元件，用于增强基因的表达水平，包括但不限于CMV的增强子、SV40增强子、EN1增强子或经过基因工程改造后的增强子变体，以及SV40 polyA加尾信号、人胰岛素基因polyA加尾信号或者WPRE(土拨鼠乙肝病毒转录后调控元件)、人源性MAR序列或经过基因工程改造后的变体。
在一优选例中，所述的载有Neurog2功能性片段的表达载体中还可以载有其他的功能性片段，所述的其他功能性片段可以是报告基因或其他具有重编程功能的转录因子功能性片段，包括但不限于Ascl1、NeuroD1等。
在一优选例中，所述的载有Neurog2功能性片段的表达载体为GFAP-AAV载体；GFAP-AAV载体载有病毒ITR序列、CMV的增强子、人GFAP的启动子、Neurog2功能性片段的编码框以及转录后调控元件WPRE等；所述的表达载体还可以载有报告基因，但报告基因在实际应用中不是必需的，如所述的GFAP-AAV表达载体自5'到3'端可依次包括以下元件：病毒ITR序列+CMV的增强子+人GFAP的启动子+Neurog2和红色荧光蛋白mCherry的编码框+转录后调控元件WPRE+病毒ITR序列+氨苄抗性基因的启动子和编码框，其中红色荧光蛋白mCherry的编码框与氨苄抗性基因的启动子和编码框不是必需的。
在一优选例中，所述的载有Neurog2功能性片段的表达载体为NG2-慢病毒载体；NG2-慢病毒载体载有病毒ITR序列、人NG2的启动子、Neurog2功能性片段的编码框以及转录后调控元件WPRE等；所述的表达载体还可以载有报告基因，但报告基因在实际应用中不是必需的，如所述的NG2-慢病毒载体自5'到3'端可依次包括以下元件：病毒ITR序列+人NG2的启动子+Neurog2和绿色荧光蛋白GFP的编码框+转录后调控元件WPRE+病毒ITR序列+氨苄抗性基因的启动子和编码框，其中绿色荧光蛋白GFP的编码框与氨苄抗性基因的启动子和编码框不是必需的。
本发明的第三方面，提供了一种包含有Neurog2功能性片段的宿主细胞。
所述的宿主细胞来源于胶质细胞，可以利用本发明第二方面所述的递送系统，在其染色体整合有编码Neurog2蛋白的多核苷酸，或在所述的宿主细胞中注入Neurog2的功能性片段，促使宿主细胞转分化为有功能的神经元。
在一优选例中，所述的宿主细胞源自体外培养的胶质细胞。
在一优选例中，所述的宿主细胞源自体内正常状态和损伤状态下的胶质细胞。
在一优选例中，所述的宿主细胞为有功能的神经元或有功能的神经元细胞群。
本发明的第四方面，提供了一种药物组合物，所述的药物组合物包括(A)本发明第一方面所述的Neurog2功能性片段；和/或(B)本发明第二方面所述的递送系统，或(C)本发明第三方面所述的宿主细胞；和(D)药学上可接受的辅料。
本发明第五方面，提供了本发明第四方面所述的药物组合物的用途，所述的药物组合物包含有Neurog2的功能性片段，可用于制备针对神经系统损伤与神经修复的药物。
所述的神经系统损伤包括脊髓损伤、癫痫、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡或不可逆的丧失等；所述的神经修复则是通过本发明第四方面所述的药物组合物，实现对神经系统损伤区域的神经元功能的恢复。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明人经过广泛而深入的研究，发现调控胶质细胞中的Neurog2基因或其蛋白表达，能够在体内或体外高效地将星形胶质细胞诱导转分化为具有正常电生理功能的神经元细胞，而且意外发现，不仅是正常状态下的星形胶质细胞，即使是处于损伤环境下的星形胶质细胞，也可以被转分化为功能性的神经元，起到神经修复的功能。特别地，来源于脊髓的胶质细胞诱导神经元可以响应外周神经系统的背根神经节(Dorsal root ganglion,DRG)的刺激，显示出经过Neurog2功能性片段诱导的神经元能够功能性的整合到脊髓环路中并发挥功能。因此，Neurog2功能性片段的诱导作用，有望成为在成人体内刺激产生新神经元细胞的有效方法，从而广泛可应用于神经系统疾病的药物开发中。
与现有技术相比，本发明主要存在以下竞争性优势：
(1)相对于其他已发现的转分化因子，Neurog2功能性片段不仅可以作用在正常环境下的胶质细胞中，还可以作用在损伤环境下的胶质细胞，突破损伤环境下胶质细胞/神经细胞的应激状态，获得具有正常电生理功能的神经连接，产生神经修复的功能。
(2)相对于其他已发现的转分化因子，Neurog2功能性片段可以特异性地将胶质细胞均一地转分化为兴奋性谷氨酸能神经元或谷氨酸能神经元群，保证了在神经系统疾病药物开发和应用的可操控性。
(3)相对于体外诱导神经元后的神经元团的移植，体内AAV介导的Neurog2诱导神经元的操作更简便，神经元诱导效率更高，诱导的神经元电生理上更为成熟，更适合用于神经系统疾病的体内再生修复。
附图说明
图1Neurog2单因子能将中脑星形胶质细胞高效率诱导成神经元。图1A，B分别显示在对照病毒AAV-mCherry(阴性对照)和病毒AAV-Neurog2/mCherry注射成年小鼠背侧中脑3天后，免疫共标显示mCherry均不与NeuN共定位。图1C显示在对照病毒AAV-mCherry注射成年小鼠背侧中脑30天后，mCherry仍不与NeuN共定位。图1D显示在病毒AAV-Neurog2/mCherry注射成年小鼠背侧中脑30天后，绝大多数mCherry与NeuN共定位。图1E显示的是不同时期神经元比率的统计图。箭与箭头分别代表mCherry+NeuN+以及mCherry+NeuN-细胞。"***"代表p<0.001。标尺：50μm。
图2Neurog2在中脑诱导的神经元通过电生理记录证实是有活性的功能性神经元。图2A显示AAV-mCherry病毒感染中脑30天后切片记录的mCherry+电生理膜参数，分别在在电流钳模式下给予梯度电压刺激记录的膜电流变化(上面)和电压钳模式下给予梯度电流刺激记录膜电压变化(下面)。图2B显示的是AAV-Neurog2/mCherry病毒感染中脑30天后切片记录的mCherry+细胞的突触后电流信号，加入阻断剂NBQX后突触后电流信号消失，洗脱后突触后电流信号重新出现。
图3显示Neurog2中脑重编程神经元为谷氨酸能神经元。图3A，B显示的是AAV-Neurog2/mCherry病毒感染成年小鼠30天后，原位组化双标显色结果。病毒信号mCherry蛋白(红色)与VGLUT2 mRNA(A,绿色)以及Gad1 mRNA(B,绿色)重叠后的信号分别为(A右边)与(B右边)。(A)中的箭指示的是mCherry+VGLUT2+双阳性的细胞。(B)中的箭头指示的是mCherry+Gad1-细胞。图3C显示的是不同递质神经元比率的统计图。细胞核被DAPI所标记。标尺：25μm。
图4Neurog2单因子能将脊髓星形胶质细胞高效率诱导成神经元。图4A，B分别显示在对照病毒AAV-mCherry和病毒AAV-Neurog2/mCherry注射成年小鼠脊髓3天后，免疫共标显示mCherry均不与NeuN共定位。图4C显示在对照病毒AAV-mCherry注射成年小鼠脊髓30天后，mCherry仍不与NeuN共定位。图4D显示在病毒AAV-Neurog2/mCherry注射成年小鼠脊髓30天后，绝大多数mCherry与NeuN共定位。图4E显示的是不同时期神经元比率的统计图。箭与箭头分别代表mCherry+NeuN+以及mCherry+NeuN-细胞。"***"代表p<0.001。标尺：50μm。
图5Neurog2在脊髓诱导神经元通过电生理记录证实是有活性的功能性神经元，并能够响应背根神经节(Dorsal root ganglion,DRG)的刺激。图5A，B显示的是脊髓切片电生理记录来源于AAV-mCherry病毒(A)和AAV-Neurog2/mCherry病毒(B)感染30天的mCherry+细胞，在电流钳或者电压钳模式下，分别给予梯度电压或者电流刺激，得到的膜电压(左)以及细胞膜电流参数(右)。图5C显示的是在DRG刺激后，脊髓背侧mCherry+诱导神经元的电生理反应。绿色箭头代表刺激的开始。
图6Neurog2单因子能将损伤脊髓星形胶质细胞高效率诱导成神经元。图6A，B分别显示在对照病毒AAV-mCherry和病毒AAV-Neurog2/mCherry注射损伤成年小鼠脊髓3天后，免疫共标显示mCherry均不与NeuN共定位。图6C显示在对照病毒AAV-mCherry注射损伤成年小鼠脊髓30天后，mCherry仍不与NeuN共定位。图6D显示在病毒AAV-Neurog2/mCherry注射损伤成年小鼠脊髓30天后，绝大多数mCherry与NeuN共定位。图6E显示的是是不同时期神经元比率的统计图。箭与箭头分别代表mCherry+NeuN+以及mCherry+NeuN-细胞。"**"代表p<0.01。标尺：50μm。
具体实施方式
Neurog2功能性片段或其促进剂
Neurog2功能性片段，为来源于哺乳动物、编码Neurogenin 2转录因子的多核苷酸或其表达蛋白片段，Neurog2为bHLH类转录因子，如来自小鼠的Neurog2分子在GenBank的ID#为11924，其蛋白序列如SEQ ID NO.:1所示；NCBI Reference Sequence:NM_009718.3；CDS序列如SEQ ID NO.:2所示；如来自人的Neurog2分子在GenBank的ID#为63973，其蛋白序列如SEQ ID NO.:3所示；NCBI Reference Sequence:NM_024019.4；CDS序列如SEQ ID NO.:4所示。
所述的Neurog2的功能性片段还可以是Neurog2蛋白序列的变体，特别地，Neurog2蛋白功能性片段的变体与SEQ ID NO.:1的序列同源性不应低于83％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于90％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于95％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于98％；或Neurog2蛋白功能性片段的变体与SEQ ID NO.:3的序列同源性不应低于83％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于90％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于95％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于98％。
所述的Neurog2的功能性片段还可以是Neurog2多核苷酸序列的变体，特别地，所述的编码Neurog2的多核酸功能性序列与SEQ ID NO.:2的序列同源性不低于80％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于90％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于95％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于98％；或所述的编码Neurog2的多核酸功能性序列与SEQ ID NO.:4的序列同源性不低于80％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于90％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于95％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于98％；
所述的Neurog2的功能性片段还可以是通过CRISPR/dCas9靶向DNA激活Neurog2基因的表达获得，或者通过CRISPR/Cas13靶向RNA提高Neurog2的表达获得；
Neurog2的表达促进剂没有特殊限制，可以为任何促进Neurog2基因或其蛋白表达和/或活性的物质，例如小分子化合物、促进性的miRNA。本领域技术人员可以根据现有的数据库对Neurog2促进剂进行筛选。应理解，基于本发明公开的Neurog2对星形胶质细胞的转分化诱导作用，本领域技术人员能够合理地预见任何对Neurog2具有促进作用的物质均会对星形胶质细胞具有转分化的诱导作用。
星形胶质细胞
星形胶质细胞，是哺乳动物脑内数量最多的一类细胞。它们执行许多功能，包括生化支撑(例如形成血-脑屏障)，为神经元提供营养，维持细胞外离子平衡，并参与脑和脊髓损伤后的修复和瘢痕形成。根据胶质丝的含量以及胞突的形状可将星形胶质细胞分为两种：纤维性星形胶质细胞(fibrous astrocyte)多分布在脑和脊髓的白质，突起细长，分支较少，胞质中含大量胶质丝；原浆性星形胶质细胞(protoplasmic astrocyte)，多分布在灰质，细胞突起粗短，分支多。
可用于本发明的星形胶质细胞没有特别限制，包括哺乳动物中枢神经系统来源的各种星形胶质细胞，例如来源于纹状体、脊髓、背侧中脑或大脑皮层，较佳地，来源于背侧中脑或脊髓。
在本发明中，各个来源的星形胶质细胞所具有的诱导转化效率均较高。通常，星形胶质细胞的特异性标志物为GFAP，灰质中的星形胶质细胞GFAP表达相对较低，但表达Acsbg1或GS。而当通过本发明方法的诱导后，这些星形胶质细胞表现出神经元细胞特有的标志物，例如NeuN。
功能性神经元
如本文所用，术语“功能性神经元”指的是在外源性Neurog2基因或蛋白存在下，由星形胶质细胞转分化而成的、具有正常神经元电生理活动的神经元细胞。通常，所述的功能性神经元细胞具有如下特性：
(a)表达神经元的标志物NeuN；
(b)能够发放动作电位并能够形成突触联系。
递送系统
可用于本发明的递送系统没有特殊限制，可以是含有Neurog2蛋白编码序列的能够进入星形胶质细胞的表达载体。例如病毒载体，其可以是任何能够利用病毒具有传送其基因组的特点，将遗传物质带入其他细胞，进行感染的病毒载体。可发生于完整活体或是细胞培养中。包括慢病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体。
递送系统还可以是新型纳米颗粒，用于装载Neurog2功能性片段，并递送到目标细胞中，如脂质体纳米颗粒、金属纳米颗粒、高分子纳米颗粒等可以携带有Neurog2功能性片段的递送系统。
递送系统还可以是包裹有Neurog2功能性片段的外泌体，或者是包裹有Neurog2功能性片段的改造红细胞或细菌。
此外，递送系统还可以结合上靶向功能的分子，如靶向星形胶质细胞的特异性单克隆抗体、多肽等，可以更好地提高Neuorog2功能性片段在星形胶质细胞上的靶向性，增加转分化效率。
诱导方法
本发明还提供了在体内将星形胶质细胞诱导转分化为功能性神经元细胞的方法。
在体内，可将含有Neurog2的递送系统施用(例如注射)到所需对象含有星形胶质细胞的部位，例如背侧中脑、大脑皮层或者脊髓，可以对未受损和受损的神经系统组织进行注射，从而诱导该神经系统特定部位中的星形胶质细胞进行转分化。
在体外，可将含有Neurog2的递送系统施用(例如注射)到体外培养的NG2胶质细胞团中，诱导有功能的神经元的体外分化，再通过移植的方式将体外培养的功能性神经元团移植到体内。
药物组合物以及给药方式
本发明还提供的一种药物组合物为含有Neurog2功能性片段的递送系统或在体外经Neurog2功能性片段诱导转分化后的功能性神经元团。
本发明药物组合物包括本发明上述的表达载体(例如病毒颗粒)、或外源性Neurog2蛋白本身，和药学上可接受的载体。
在本发明的药物组合物，通常含有1010-1013PFU/ml的AAV病毒颗粒，较佳地1011-1013PFU/ml的AAV病毒颗粒，更佳地1010-1012PFU/ml的AAV病毒颗粒。
“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
通常，将所述表达载体和药学上可接受的载体混合后，即可获得的本发明的药物组合物。
本发明所述的组合物的给药方式没有特别限制，代表性的例子包括(但并不限于)：静脉注射、皮下注射、脑部注射、鞘内注射和脊髓注射等。
应用
本发明Neurog2功能性片段可用于制备诱导星形胶质细胞产生功能性神经元的药物，从而将新诱导的神经元应用于各种由于神经元数量减少、细胞衰退、凋亡或神经元功能下降相关的疾病。其中，所述的神经系统相关疾病包括脊髓损伤、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡等。
本发明有益效果
本发明将单个转录因子Neurog2能够将成年小鼠背侧中脑以及脊髓的星形胶质细胞转分化为功能性的神经元。这些诱导的神经元表达神经元的标志分子，能够发放动作电位，并能够接受其他神经元的突触传入建立突触联系。因此，该方法有望成为在成人体内刺激产生新神经元细胞的有效方法，从而广泛应用于神经系统疾病的治疗，例如神经退行性病变、中枢神经创伤性疾病等等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
通用方法
NG2细胞培养
对于NG2细胞的制备培养，取出出生后3-5天小鼠的皮质组织，并用0.25％的胰酶消化15分钟。吹散的细胞置于含10％血清的DMEM/F12配液中培养7-9天。然后振荡烧瓶并收集上清，离心重悬细胞并接种在多聚-D-赖氨酸(Sigma)包被的盖玻片上，在37℃下在含5％CO2的培养箱中含有N2补充剂(STEMCELLTM)、10ng/ml血小板衍生生长因子(PDGF，Invitrogen)、10ng/mL表皮生长因子(EGF，Invitrogen)和10ng/mL成纤维细胞生长因子2(FGF2,Invitrogen)的无血清DMEM培养基(Gibco)中培养细胞3天。
免疫显色
培养细胞的免疫显色参照“Direct conversion of fibroblasts to functionalneurons by defined factors”(Vierbuchen,T.et al.Nature 463,1035-1041(2010)).组织切片的免疫显色结合原位杂交及免疫显色的双标实验参照已发表的方法进行。免疫显色用到的一抗包括：mouse anti-GFAP(Millipore,1:1,000),mouse anti-NeuN(Millipore,1:100),anti-Dsred(Clontech,1:500),mouse anti-Dsred(Santa Cruz,1:100)，rabbitanti-Acsbg1(Abcam,1:100),rabbit anti-NG2(Millipore,1:200),rabbit anti-Iba1(Wako,1:500),mouse anti-CNPase(Abcam,1:500),mouse anti-O4(Millipore,1:500)。FITC-,Cy3-以及Cy5-偶联的二抗购自Jackson Immunoresearch。
AAV病毒的立体定位注射
AAV病毒参照小鼠脑图谱进行。注射病毒后，在不同时间点收集背部中脑、脊髓做免疫显色或脑片记录。完整脊髓以及损伤脊髓病毒注射浓度，速度与每针注射量与脑区一致，在脊髓是以30°夹角的方位注射的。
诱导神经元的移植
移植所使用的小鼠为七周的NOD-scid小鼠。Accutase消化诱导4-6天的细胞，离心去除上清使得细胞浓缩后密度大约为2×105cells/μl，每只小鼠大脑移植2μl即总共4×105细胞。移植后2-4周进行组化以及电生理检测。
脊髓损伤检测模型
脊髓胸段损伤后导致感觉传入的缺失促使脑干的下行抑制系统的抑制作用减弱导致尾巴对外界刺激过度敏感。我们利用甩尾实验模型，通过测量两组小鼠尾巴在48℃以及52℃的热刺激下反应延迟时间来检测小鼠的感觉能力。根据BMS标准对小鼠运动功能进行评分。试验方法参照Basso Mouse Scale for locomotion detects differences inrecovery after spinal cord injury in five common mouse strains.J Neurotrauma,2006.23(5):p.635-59.
Open field旷场实验
被用于动物焦虑水平的评估。通常认为，小鼠在旷场中心区域的活动与焦虑程度相关，正常小鼠会频繁穿梭旷场中心区域(Zone Crossing)，而焦虑小鼠倾向于更多在广场四周运动(Peripheral)。实验方法参照The open field as a paradigm to measure theeffects of drugs on anxiety-like behaviors:a review.European Journal ofPharmacology,2003.463(1-3):p.3-33.
实施例1 Neurog2在体外将NG2胶质细胞转分化为功能性神经元
(1)质粒构建与病毒感染
在FUGW–IRES–EGFP的载体模板(载体信息参考文献Efficient transfer,integration,and sustained long-term expression of the transgene in adult ratbrains injected with a lentiviral vector.Proc Natl Acad Sci U S A 93:11382–11388)上克隆人NG2启动子(SEQ ID No.:5)替换CAG启动子，再将源自人Neurog2基因的CDS(SEQ ID No.:4)片段构建到该慢病毒载体上，以生成hNG2-hNeurog2-IRES-EGFP慢病毒质粒。慢病毒的包装参照文献“Production and purification of lentiviral vectors”(Tiscornia,G.,Singer,O.&Verma,I.M.Nat.Protoc.1,241-245(2006))。
NG2细胞铺板培养24小时后加入慢病毒，感染24小时后更换培养基：DMEM/F12，B27，Glutamax和青霉素/链霉素。感染6-7后，每三天在培养基中加入脑源性神经营养因子(BDNF；PeproTech公司，20ng/ml)。
(2)Neurog将NG2细胞转分化为神经元
培养的小鼠NG2细胞大多数对NG2胶质细胞标记物NG2为免疫阳性，少量的细胞表达少突胶质细胞的标志分子O4和CNPase，没有检测到神经元标志分子Tuj1以及干细胞标志分子Sox2和Oct4的表达。
NG2细胞转染hNG2-Neurog2-IRES-GFP慢病毒10天后，大部分NG2细胞呈现典型的神经元形态，表达神经元的标志分子Tuj1。感染慢病毒21天后，诱导细胞细胞还表达成熟神经元的标志分子NeuN和MAP2。电生理记录表明诱导的神经元能产生动作电位，而且绝大多数的诱导神经元细胞能记录到自发的突触后电流，这说明这些神经元能形成功能性突触。
(3)NG2细胞诱导的神经元移植到体内可以存活
体外诱导得到的转分化神经元能否在体内存活并发挥功能是其是否可用于疾病治疗的关键。Neurog2在NG2细胞诱导的神经元移植到大脑皮层后，两周进行免疫组化实验，我们发现移植的细胞能够依附在皮层边缘，且部分细胞能够将神经突起伸向皮层更深处。免疫荧光共定位实验结果显示移植的细胞有一部分表达神经元标志分子Tuj1，说明诱导的神经元能够存活并表达神经元特异的标志分子。
实施例2 Neurog2在体内将成年背侧中脑星形胶质细胞转分化为神经元
(1)腺相关病毒质粒构建与病毒感染
在AAV–FLEX–Arch–GFP(Addgene，#22222)的载体模板上克隆GFAP启动子(SEQ IDNo.:5)替换CAG启动子并保留CMV增强子，再用mCherry的编码框取代GFP后得到AAV-mCherry质粒(对照组)。将源自小鼠Neurog2基因的CDS(SEQ ID No.:2,NCBI:NM_009718.3)克隆进AAV-mCherry质粒后得到AAV-mNeurog2/mCherry质粒，目的基因在GFAP启动子作用下可以特异靶向星形胶质细胞。
(2)Neurog2将星形胶质细胞转分化为神经元
将病毒AAV-mCherry或AAV-mNeurog2/mCherry注射到成年野生型小鼠的一侧顶盖，然后在不同的时间点收集脑组织样品。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图1A)，还是病毒AAV-Neurog2/mCherry(图1B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图1C)。然而，在注射病毒AAV-Neurog2/mCherry的小鼠中，绝大多数mCherry与NeuN共定位(88.2±6.3％)(图1D、图1E)。说明Neurog2成功将星形胶质细胞诱导为神经元。
(3)经诱导的神经元为有功能的神经元
为了证明诱导神经元是有功能的活性神经元，我们进行了电生理记录。我们发现AAV-mCherry病毒感染中脑30天后切片记录的mCherry+细胞并不能产生动作电位(图2A)，这说明AAV-mCHerry病毒体内靶向了星形胶质细胞没有改变它们的电生理特性。而在记录Neurog2/mCherry病毒感染的脑切片时，我们发现20个被记录细胞中，19个细胞在电压钳模式下表现出了内向Na+电流以及外向K+电流(19/20)。而且19个被记录的细胞能够发放多个动作电位(19/20)。重要的是，17个被记录的细胞能产生兴奋性突触后电流(Excitatorypostsynaptic currents,EPSCs)(17/20)(图2B)，加入阻断剂NBQX后突触后电流信号消失，洗脱后突触后电流信号重新出现。这说明Neurog2诱导的神经元整合到神经环路中建立了突触联系，是功能性的神经元。
(4)经诱导的神经元均为兴奋性神经元
我们对Neurog2诱导的神经元递质属性进行了鉴定。通过使用原位杂交与免疫组化双标的方法对VGLUT2/Gad1 mRNA与mCherry蛋白进行染色。实验结果显示，AAV-Neurog2/mCherry病毒感染30天后能将中脑星形胶质细胞重编程为VGLUT2+兴奋性神经元(图3A，C)，而非Gad1+抑制性神经细胞(图3B，C)。因此，我们证实Neurog2单因子可以将中脑星形胶质细胞在体重编程为谷氨酸能神经元。
实施例3 Neurog2在体内将成年小鼠正常脊髓星形胶质细胞转分化成神经元
(1)质粒构建与病毒感染
利用实施例2所述的腺相关病毒载体AAV-mCherry质粒，将源自人Neurog2基因的CDS(SEQ ID No.:4,NCBI:NM_024019.4)克隆进AAV-mCherry后得到AAV-hNeurog2/mCherry质粒。
(2)Neurog2将星形胶质细胞转分化为神经元
我们将AAV-mCherry病毒以及AAV-hNeurog2/mCherry病毒注射到成年小鼠胸段(Thoracic，T8-T10)脊髓背侧，3天后发现两个病毒都能特异性感染星形胶质细胞。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图4A)，还是病毒AAV-Neurog2/mCherry(图4B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图4C)。然而，在注射病毒AAV-Neurog2/mCherry的小鼠中，绝大多数mCherry与NeuN共定位(图4D、图4E)。说明Neurog2成功将脊髓星形胶质细胞诱导为神经元。
(3)经诱导的神经元为有功能的神经元
我们对Neurog2在脊髓重编程神经元进行电生理特性分析。通过脑片全细胞记录的方法发现，AAV-mCherry病毒感染30天后，mCherry+细胞在给予胞内梯度电流刺激下不能产生动作电位，而且在电压钳模式下刺激也不能产生内向电流(图5A)。这些特征与报道的星形胶质细胞一致。而AAV-Neurog2/mCherry病毒感染30天后，大部分mCherry+细胞在梯度电流刺激下能够发放多个动作电位，而且在电压钳模式刺激下能够产生内向Na+电流以及外向K+电流(图5B)。我们进一步检测Neurog2重编程神经元是否整合到现有的神经环路。我们将脊髓连同DRG部分一起取出进行电生理检测。当我们刺激背根神经节DRG时，能够检测到脊髓背侧诱导的神经元产生的兴奋性突触后电流(Excitatory postsynapticcurrents,EPSC)和兴奋性突触后电位(Excitatory postsynaptic potential,EPSP)信号(图5C)，这说明Neurog2重编程神经元Neurog2在完整脊髓重编程神经元能够整合到神经环路并发挥潜在的功能。
实施例4 Neurog2在体内将成年小鼠损伤脊髓星形胶质细胞转分化成神经元
脊髓损伤(Spinal cord injury，SCI)是一类中枢神经受损疾病，伴随着脊髓神经元的死亡以及胶质疤痕的形成。在体神经元重编程将星形胶质细胞转化为神经元，有可能可以缓解SCI造成的伤害，有望成为新的治疗手段。
(1)Neurog2将星形胶质细胞转分化为神经元
我们尝试了使用Neurog2将损伤环境下的胶质细胞重编程为神经元。首先，构造了小鼠T8-T10脊髓全断模型(参照McDonough A,Monterrubio A,Ariza J,et al.CalibratedForceps Model of Spinal Cord Compression Injury.Jove-Journal of VisualizedExperiments 2015.的方法),损伤后立即将AAV-mCherry病毒以及AAV-mNeurog2/mCherry(实施例2)或AAV-hNeurog2/mCherry(实施例3)分别注射到损伤脊髓两侧。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图6A)，还是病毒AAV-mNeurog2/mCherry(图6B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图6C)。然而，在注射病毒AAV-mNeurog2/mCherry的小鼠中，能发现部分mCherry与NeuN共定位(图6D、图6E)。说明Neurog2成功将损伤脊髓星形胶质细胞诱导为神经元。同样地，由于载有人源的Neurog2的腺相关病毒载体质粒AAV-hNeurog2/mCherry也能成功地将损伤的脊髓星形胶质细胞诱导为神经元。
(2)脊髓损伤模型的修复
由于脊髓损伤后的Neurog2重编程神经元具备电生理特征，而且能够接受外来信号输入。因此，我们使用了多种动物模型来评测脊髓损伤小鼠经过神经元诱导后的神经修复能力。应用脊髓损伤检测模型，我们发现Neurog2重编程神经元对脊髓损伤小鼠感觉功能以及运动功能的恢复有很大帮助。
我们还使用小鼠广场实验来评估小鼠损伤后的焦虑程度，来检测经Neurog2重编程的神经元能否能减缓部分脊髓损伤小鼠焦虑水平，由于脊髓损伤造成的急/慢性疼痛，特别是慢性疼痛通常能导致焦虑。脊髓损伤的小鼠模型，通常伴随着不同程度的焦虑，我们发现，在经Neurog2重编程神经元对脊髓损伤小鼠的焦虑的也实现了极大的缓解。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述的实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
SEQUENCE LISTING
<110> 再康医药科技（上海）有限公司
<120> 一种诱导胶质细胞转分化为功能性神经元的方法及其应用
<130> 2020.05.08
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 263
<212> PRT
<213> Mus musculus
<400> 1
Met Phe Val Lys Ser Glu Thr Leu Glu Leu Lys Glu Glu Glu Glu Val
1 5 10 15
Leu Met Leu Leu Gly Ser Ala Ser Pro Ala Ser Ala Thr Leu Thr Pro
20 25 30
Met Ser Ser Ser Ala Asp Glu Glu Glu Asp Glu Glu Leu Arg Arg Pro
35 40 45
Gly Ser Ala Arg Gly Gln Arg Gly Ala Glu Ala Glu Gln Gly Val Gln
50 55 60
Gly Ser Pro Ala Ser Gly Ala Gly Gly Cys Arg Pro Gly Arg Leu Leu
65 70 75 80
Gly Leu Met His Glu Cys Lys Arg Arg Pro Ser Arg Ser Arg Ala Val
85 90 95
Ser Arg Gly Ala Lys Thr Ala Glu Thr Val Gln Arg Ile Lys Lys Thr
100 105 110
Arg Arg Leu Lys Ala Asn Asn Arg Glu Arg Asn Arg Met His Asn Leu
115 120 125
Asn Ala Ala Leu Asp Ala Leu Arg Glu Val Leu Pro Thr Phe Pro Glu
130 135 140
Asp Ala Lys Leu Thr Lys Ile Glu Thr Leu Arg Phe Ala His Asn Tyr
145 150 155 160
Ile Trp Ala Leu Thr Glu Thr Leu Arg Leu Ala Asp His Cys Ala Gly
165 170 175
Ala Gly Gly Leu Gln Gly Ala Leu Phe Thr Glu Ala Val Leu Leu Ser
180 185 190
Pro Gly Ala Ala Leu Gly Ala Ser Gly Asp Ser Pro Ser Pro Pro Ser
195 200 205
Ser Trp Ser Cys Thr Asn Ser Pro Ala Ser Ser Ser Asn Ser Thr Ser
210 215 220
Pro Tyr Ser Cys Thr Leu Ser Pro Ala Ser Pro Gly Ser Asp Val Asp
225 230 235 240
Tyr Trp Gln Pro Pro Pro Pro Glu Lys His Arg Tyr Ala Pro His Leu
245 250 255
Pro Leu Ala Arg Asp Cys Ile
260
<210> 2
<211> 792
<212> DNA
<213> Mus musculus
<400> 2
atgttcgtca aatctgagac tctggagttg aaggaggaag aggaggtact gatgctgctg 60
ggctcggctt ccccggcctc ggcgaccctg accccgatgt cctccagcgc ggacgaggag 120
gaggacgagg agctgcgccg gccgggctcc gcgcgtgggc agcgtggagc ggaagccgag 180
cagggggtgc agggcagtcc ggcgtcgggt gccgggggtt gccggccagg gcggctgctg 240
ggcctgatgc acgagtgcaa gcgtcgcccg tcgcgctcac gggccgtctc ccgaggtgcc 300
aagacggcgg agacggtgca gcgcatcaag aagacccgca ggctcaaggc caacaaccgc 360
gagcgcaacc gcatgcacaa cctaaacgcc gcgctggacg cgctgcgcga ggtgctgccc 420
accttccccg aggatgccaa gctcacgaag atcgagacgc tgcgcttcgc ccacaattac 480
atctgggcgc tcaccgagac tctgcgcctg gcggaccact gcgccggcgc cggtggcctc 540
cagggggcgc tcttcacgga ggcggtgctc ctgagcccgg gagctgcgct cggcgccagc 600
ggggacagcc cttctccacc ttcctcctgg agctgcacca acagcccggc gtcatcctcc 660
aactccacgt ccccatacag ctgcacttta tcgcccgcta gccccgggtc agacgtggac 720
tactggcagc ccccacctcc ggagaagcat cgttatgcgc ctcacctgcc cctcgccagg 780
gactgtatct ag 792
<210> 3
<211> 272
<212> PRT
<213> Homo sapiens
<400> 3
Met Phe Val Lys Ser Glu Thr Leu Glu Leu Lys Glu Glu Glu Asp Val
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Leu Val Leu Leu Gly Ser Ala Ser Pro Ala Leu Ala Ala Leu Thr Pro
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35 40 45
Gly Gly Ala Arg Arg Gln Arg Gly Ala Glu Ala Gly Gln Gly Ala Arg
50 55 60
Gly Gly Val Ala Ala Gly Ala Glu Gly Cys Arg Pro Ala Arg Leu Leu
65 70 75 80
Gly Leu Val His Asp Cys Lys Arg Arg Pro Ser Arg Ala Arg Ala Val
85 90 95
Ser Arg Gly Ala Lys Thr Ala Glu Thr Val Gln Arg Ile Lys Lys Thr
100 105 110
Arg Arg Leu Lys Ala Asn Asn Arg Glu Arg Asn Arg Met His Asn Leu
115 120 125
Asn Ala Ala Leu Asp Ala Leu Arg Glu Val Leu Pro Thr Phe Pro Glu
130 135 140
Asp Ala Lys Leu Thr Lys Ile Glu Thr Leu Arg Phe Ala His Asn Tyr
145 150 155 160
Ile Trp Ala Leu Thr Glu Thr Leu Arg Leu Ala Asp His Cys Gly Gly
165 170 175
Gly Gly Gly Gly Leu Pro Gly Ala Leu Phe Ser Glu Ala Val Leu Leu
180 185 190
Ser Pro Gly Gly Ala Ser Ala Ala Leu Ser Ser Ser Gly Asp Ser Pro
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Ser Pro Ala Ser Thr Trp Ser Cys Thr Asn Ser Pro Ala Pro Ser Ser
210 215 220
Ser Val Ser Ser Asn Ser Thr Ser Pro Tyr Ser Cys Thr Leu Ser Pro
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Ala Ser Pro Ala Gly Ser Asp Met Asp Tyr Trp Gln Pro Pro Pro Pro
245 250 255
Asp Lys His Arg Tyr Ala Pro His Leu Pro Ile Ala Arg Asp Cys Ile
260 265 270
<210> 4
<211> 819
<212> DNA
<213> Homo sapiens
<400> 4
atgttcgtca aatccgagac cttggagttg aaggaggaag aggacgtgtt agtgctgctc 60
ggatcggcct cccccgcctt ggcggccctg accccgctgt catccagcgc cgacgaagaa 120
gaggaggagg agccgggcgc gtcaggcggg gcgcgtcggc agcgcggggc tgaggccggg 180
cagggggcgc ggggcggcgt ggctgcgggt gcggagggct gccggcccgc acggctgctg 240
ggtctggtac acgattgcaa acggcgccct tcccgggcgc gggccgtctc ccgaggcgcc 300
aagacggccg agacggtgca gcgcatcaag aagacccgta gactgaaggc caacaaccgc 360
gagcgaaacc gcatgcacaa cctcaacgcg gcactggacg cgctgcgcga ggtgctcccc 420
acgttccccg aggacgccaa gctcaccaag atcgagaccc tgcgcttcgc ccacaactac 480
atctgggcac tcaccgagac cctgcgcctg gcggatcact gcgggggcgg cggcgggggc 540
ctgccggggg cgctcttctc cgaggcagtg ttgctgagcc cgggaggagc cagcgccgcc 600
ctgagcagca gcggagacag cccctcgccc gcctccacgt ggagttgcac caacagcccc 660
gcgccgtcct cctccgtgtc ctccaattcc acctccccct acagctgcac tttatcgccc 720
gccagcccgg ccgggtcaga catggactat tggcagcccc cacctcccga caagcaccgc 780
tatgcacctc acctccccat agccagggat tgtatctag 819
<210> 5
<211> 1387
<212> DNA
<213> Homo sapiens
<400> 5
ctctggtttc aagaccaata ctcataaccc ccacatggac caggcaccat cacacctgag 60
cactgcactt agggtcaaag acctggcccc acatctcagc agctatgtag actagctcca 120
gtcccttaat ctctctcagc ctcagtttct tcatctgcaa aacaggtctc agtttcgttg 180
caaagtatga agtgctgggc tgttactggt caaagggaag agctgggaag agggtgcaag 240
gtggggttgg gctggagatg ggctggagca gatagatgga gggacctgaa tggaggaagt 300
aaaccaaggc ccggtaacat tgggactgga cagagaacac gcagatcctc taggcaccgg 360
aagctaagta acattgccct ttctcctcct gtttgggact aggctgatgt tgctgcctgg 420
aagggagcca gcagaagggc cccagcctga agctgttagg tagaagccaa atccagggcc 480
agatttccag gaggcagcct cgggaagttg aaacacccgg attcaggggt caggaggcct 540
gggcttctgg caccaaacgg ccagggacct actttccacc tggagtcttg taagagccac 600
tttcagcttg agctgcactt tcgtcctcca tgaaatgggg gaggggatgc tcctcaccca 660
ccttgcaagg ttattttgag gcaaatgtca tggcgggact gagaattctt ctgccctgcg 720
aggaaatcca gacatctctc ccttacagac agggagactg aggtgaggcc cttccaggca 780
gagaaggtca ctgttgcagc catgggcagt gccccacagg acctcgggtg gtgcctctgg 840
agtctggaga agttcctagg ggacctccga ggcaaagcag cccaaaagcc gcctgtgagg 900
gtggctggtg tctgtccttc ctcctaaggc tggagtgtgc ctgtggaggg gtctcctgaa 960
ctcccgcaaa ggcagaaagg agggaagtag gggctgggac agttcatgcc tcctccctga 1020
gggggtctcc cgggctcggc tcttggggcc agagttcagg gtgtctgggc ctctctatga 1080
ctttgttcta agtctttagg gtggggctgg ggtctggccc agctgcaagg gccccctcac 1140
ccctgcccca gagaggaaca gccccgcacg ggccctttaa gaaggttgag ggtgggggca 1200
ggtgggggag tccaagcctg aaacccgagc gggcgcgcgg gtctgcgcct gccccgcccc 1260
cggagttaag tgcgcggaca cccggagccg gcccgcgccc aggagcagag ccgcgctcgc 1320
tccactcagc tcccagctcc caggactccg ctggctcctc gcaagtcctg ccgcccagcc 1380
cgccggg 1387
<210> 6
<211> 2209
<212> DNA
<213> Homo sapiens
<400> 6
taatcccacc tccctctctg tgctgggact cacagaggga gacctcagga ggcagtctgt 60
ccatcacatg tccaaatgca gagcataccc tgggctgggc gcagtggcgc acaactgtaa 120
ttccagcact ttgggaggct gatgtggaag gatcacttga gcccagaagt tctagaccag 180
cctgggcaac atggcaagac cctatctcta caaaaaaagt taaaaaatca gccacgtgtg 240
gtgacacaca cctgtagtcc cagctattca ggaggctgag gtgaggggat cacttaaggc 300
tgggaggttg aggctgcagt gagtcgtggt tgcgccactg cactccagcc tgggcaacag 360
tgagaccctg tctcaaaaga caaaaaaaaa aaaaaaaaaa aaagaacata tcctggtgtg 420
gagtagggga cgctgctctg acagaggctc gggggcctga gctggctctg tgagctgggg 480
aggaggcaga cagccaggcc ttgtctgcaa gcagacctgg cagcattggg ctggccgccc 540
cccagggcct cctcttcatg cccagtgaat gactcacctt ggcacagaca caatgttcgg 600
ggtgggcaca gtgcctgctt cccgccgcac cccagccccc ctcaaatgcc ttccgagaag 660
cccattgagc agggggcttg cattgcaccc cagcctgaca gcctggcatc ttgggataaa 720
agcagcacag ccccctaggg gctgcccttg ctgtgtggcg ccaccggcgg tggagaacaa 780
ggctctattc agcctgtgcc caggaaaggg gatcagggga tgcccaggca tggacagtgg 840
gtggcagggg gggagaggag ggctgtctgc ttcccagaag tccaaggaca caaatgggtg 900
aggggactgg gcagggttct gaccctgtgg gaccagagtg gagggcgtag atggacctga 960
agtctccagg gacaacaggg cccaggtctc aggctcctag ttgggcccag tggctccagc 1020
gtttccaaac ccatccatcc ccagaggttc ttcccatctc tccaggctga tgtgtgggaa 1080
ctcgaggaaa taaatctcca gtgggagacg gaggggtggc cagggaaacg gggcgctgca 1140
ggaataaaga cgagccagca cagccagctc atgtgtaacg gctttgtgga gctgtcaagg 1200
cctggtctct gggagagagg cacagggagg ccagacaagg aaggggtgac ctggagggac 1260
agatccaggg gctaaagtcc tgataaggca agagagtgcc ggccccctct tgccctatca 1320
ggacctccac tgccacatag aggccatgat tgacccttag acaaagggct ggtgtccaat 1380
cccagccccc agccccagaa ctccagggaa tgaatgggca gagagcagga atgtgggaca 1440
tctgtgttca agggaaggac tccaggagtc tgctgggaat gaggcctagt aggaaatgag 1500
gtggcccttg agggtacaga acaggttcat tcttcgccaa attcccagca ccttgcaggc 1560
acttacagct gagtgagata atgcctgggt tatgaaatca aaaagttgga aagcaggtca 1620
gaggtcatct ggtacagccc ttccttccct tttttttttt tttttttgtg agacaaggtc 1680
tctctctgtt gcccaggctg gagtggcgca aacacagctc actgcagcct caacctactg 1740
ggctcaagca atcctccagc ctcagcctcc caaagtgctg ggattacaag catgagccac 1800
cccactcagc cctttccttc ctttttaatt gatgcataat aattgtaagt attcatcatg 1860
gtccaaccaa ccctttcttg acccaccttc ctagagagag ggtcctcttg cttcagcggt 1920
cagggcccca gacccatggt ctggctccag gtaccacctg cctcatgcag gagttggcgt 1980
gcccaggaag ctctgcctct gggcacagtg acctcagtgg ggtgagggga gctctcccca 2040
tagctgggct gcggcccaac cccaccccct caggctatgc cagggggtgt tgccaggggc 2100
acccgggcat cgccagtcta gcccactcct tcataaagcc ctcgcatccc aggagcgagc 2160
agagccagag caggttggag aggagacgca tcacctccgc tgctcgcgg 2209
Claims (10)
1.一种Neurog2功能性片段的用途，其特征在于，(i)用于制备神经诱导胶质细胞形成功能性神经元细胞的药物组合物；和/或(ii)用于制备针对神经系统疾病的药物组合物。
2.如权利要求1所述的用途，其特征在于，所述的胶质细胞选自人或非人哺乳动物的星形胶质细胞、NG2胶质细胞、少突胶质细胞或小胶质细胞；优选地，所述的胶质细胞为星形胶质细胞。
3.如权利要求2所述的用途，其特征在于，所述的胶质细胞来源于脊髓、背侧中脑或大脑皮层；较佳地，所述的胶质细胞来源于脊髓和背侧中脑；其中，所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
4.如权利要求1所述的用途，其特征在于，所述的Neurog2功能性片段为具有功能性的Neurog2蛋白或编码Neurog2的核酸序列：
其中，所述的Neurog2蛋白与SEQ ID NO.:1或SEQ ID NO.:3的序列同源性不低于83％；更优地，所述的Neurog2蛋白与SEQ ID NO.:1的序列同源性不低于90％；最优地，所述的Neurog2蛋白与SEQ ID NO.:1的序列同源性不低于95％；
所述的编码Neurog2的核酸序列与SEQ ID NO.:2或SEQ ID NO.:4的序列同源性不低于80％；更优地，所述的编码Neurog2的核酸序列与SEQ ID NO.:2的序列同源性不低于90％；最优地，所述的编码Neurog2的核酸序列与SEQ ID NO.:2的序列同源性不低于95％。
5.一种载有Neurog2功能性片段的递送系统，其特征在于，所述的递送系统应用于体内或应用于体外，诱导胶质细胞转分化为功能性神经元；所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
6.如权利要求5所述的递送系统，其特征在于，所述的递送系统为病毒载体，选自腺相关病毒载体、腺病毒载体、逆转录病毒载体或慢病毒载体。
7.如权利要求6所述的递送系统，其特征在于，所述病毒载体至少包含胶质细胞特异性的启动子和Neurog2功能性片段。
8.如权利要求7所述的递送系统，其特征在于，所述的病毒载体的启动子选自GFAP启动子、NG2启动子、Aldh1L1启动子、IBA1启动子、CNP启动子、LCN2启动子或经过基因工程改造后的启动子变体；优选地，所述的病毒载体为GFAP-AAV载体或NG2-慢病毒载体。
9.一种载有Neurog2功能性片段的宿主细胞，其特征在于，所述的宿主细胞来源于体外培养的胶质细胞或体内正常状态下或损伤状态下的胶质细胞的转分化，并形成有功能的神经元或有功能的神经元细胞群；
其中，所述有功能的神经元具有以下特征：
(a)表达神经元的标志物NeuN；
(b)发放动作电位并形成突触联系；
所述有功能的神经元细胞群具有以下特征：
(a)至少50％的神经元细胞，优选至少60％、70％、80％、90％或100％的神经元细胞表达成熟神经元的标志物NeuN；
(b)发放动作电位并形成突触联系。
10.如权利要求5-8的任一递送系统或如权利要求9所述的宿主细胞的应用，其特征在于，所述的递送系统或所述的宿主细胞用于制备针对神经系统损伤与神经修复的药物组合物，所述的药物组合物还包含有药学上可接受的载体。
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| CN202010399397.1A CN113652402A (zh) | 2020-05-12 | 2020-05-12 | 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 |
| PCT/CN2021/092851 WO2021228050A1 (zh) | 2020-05-12 | 2021-05-10 | 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 |
| US17/924,685 US20230201304A1 (en) | 2020-05-12 | 2021-05-10 | Method for inducing glial cells transdifferentiation into functional neurons, and application thereof |
| JP2022569179A JP2023524900A (ja) | 2020-05-12 | 2021-05-10 | 機能性ニューロンへのグリア細胞の分化転換を誘導するための方法、及びその用途 |
| CN202180034333.4A CN115605583A (zh) | 2020-05-12 | 2021-05-10 | 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 |
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| CN114703196A (zh) * | 2022-05-16 | 2022-07-05 | 中国科学技术大学 | 诱导神经元分化的rna组合物、分化方法及其应用 |
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2021
- 2021-05-10 US US17/924,685 patent/US20230201304A1/en active Pending
- 2021-05-10 WO PCT/CN2021/092851 patent/WO2021228050A1/zh unknown
- 2021-05-10 JP JP2022569179A patent/JP2023524900A/ja active Pending
- 2021-05-10 EP EP21805293.4A patent/EP4151724A1/en active Pending
- 2021-05-10 CN CN202180034333.4A patent/CN115605583A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703196A (zh) * | 2022-05-16 | 2022-07-05 | 中国科学技术大学 | 诱导神经元分化的rna组合物、分化方法及其应用 |
| CN114703196B (zh) * | 2022-05-16 | 2024-03-29 | 中国科学技术大学 | 诱导神经元分化的rna组合物、分化方法及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP4151724A1 (en) | 2023-03-22 |
| JP2023524900A (ja) | 2023-06-13 |
| US20230201304A1 (en) | 2023-06-29 |
| WO2021228050A1 (zh) | 2021-11-18 |
| CN115605583A (zh) | 2023-01-13 |
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