CN110396499A - 一种诱导神经干细胞的方法及其应用 - Google Patents
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Abstract
本发明公开了一种诱导神经干细胞的方法及组合物,利用所述方法和组合物可将外周血单个核细胞诱导形成神经干细胞。所述神经干细胞能表达神经干细胞相关基因,能分化出神经元,星形胶质细胞及少突胶质细胞。上述神经干细胞体外诱导分化形成的多巴胺能神经前体细胞,移植入PD小鼠模型纹状体内无肿瘤形成,可改善小鼠帕金森病模型的行为学,并能延缓帕金森疾病的进程。本发明提供的神经干细胞诱导及分化方法操作简单快速,创伤性小,安全性良好,有望用于治疗帕金森病。
Description
技术领域
本发明涉及生物医药领域及神经干细胞技术领域,具体涉及一种利用非整合可升温灭活的仙台病毒载体诱导神经干细胞的方法及其定向分化的多巴胺能神经前体细胞在帕金森病细胞移植治疗的用途。
背景技术
干细胞移植为许多神经系统疾病的治疗带来希望,如帕金森病、肌萎缩性脊髓侧索硬化症、脑及脊髓外伤等。研究表明,使用胚胎干细胞来源的神经干细胞治疗以上疾病有一定作用,但胚胎干细胞的致瘤性,异体移植的免疫排斥以及伦理学争议限制其应用。由人成纤维细胞获得诱导多能干细胞 (iPSCs),并将其分化成神经干细胞用于移植治疗神经系统病变。然而研究发现,iPSCs用于临床治疗还面临很多问题,如致瘤性,定向分化效率低,安全性问题。近年来,将体细胞直接重编程为诱导神经干细胞(induced neural stemcells iNSCs),而不经过iPSCs阶段,从而减少其致瘤性、提高分化效率,成为现在干细胞研究领域中的研究热点。
在诱导神经干细胞(iNSCs)的研究中,Ring等用逆转录病毒载体携带 sox2转染人成纤维细胞,6-10天后就出现神经干细胞克隆,但是逆转录病毒基因序列能整合在宿主细胞的基因序列中导致宿主细胞基因突变。裴端卿等使用非整合质粒载体oriP/EBNA携带Oct4,Sox2,Sv40LT,Klf4和 microRNA302-367电转人尿液中提取的活细胞,获得能表Sox1,Sox2,Pax6的神经干细胞,诱导的神经干细胞能分化成星形胶质细胞,多巴胺能神经元。张素春等利用仙台病毒携带外源Oct4,Sox2,Klf4,c-Myc基因,转染人成纤维细胞后在含有LIF,SB431541,CHIR99021的神经干细胞培养基中培养13天后有神经干细胞克隆出现,这种诱导的神经干细胞可以分化出神经元,星形细胞,少突胶质细胞。但是,从人体获取成纤维细胞是有创伤的操作,成纤维细胞在转染前需在体外培养较长时间,而且,老年患者成纤维细胞不容易转染成功。
由此可见,现有技术中采用病毒载体或质粒载体诱导人成纤维细胞或尿液中提取的细胞时,存在如下问题。1.获取成纤维细胞创伤较大,体外培养成纤维细胞较难。2.细胞来源容易受到污染,如从尿液中获取细胞在操作过程中容易污染,特别是从病房获得尿液,更加容易污染。3.整合性病毒载体可能引起宿主细胞的基因突变。
帕金森病的细胞治疗近20年来一直受到广大医务工作者及生物学领域学者的关注,但目前没有报道用诱导神经干细胞分化而来的多巴胺能神经前体细胞进行移植治疗。
发明内容
本发明的目的是提供一种利用非整合载体将外周血单个核细胞诱导成为神经干细胞的技术,将神经干细胞定向分化为多巴胺能神经前体细胞治疗帕金森病,可改善帕金森病动物模型的行为学,可以对帕金森病进行细胞移植治疗。
本发明的第一方面提供了一种诱导神经干细胞的方法,所述方法包含以下步骤:(1)从外周血中提取单个核细胞并在单个核细胞培养基中扩增单个核细胞;(2)利用携带基因OCT4,SOX2,c-MYC,KLF-4的仙台病毒载体转染扩增后的单个核细胞;(3)将转染的单个核细胞接种到用Matrigel包被的细胞培养板上在神经干细胞培养基中培养,继续培养直至出现神经干细胞克隆;(4)取出神经干细胞克隆,进行神经干细胞扩增,期间将神经干细胞高温培养,获得仙台病毒被灭活的神经干细胞。
在一优选例中,所述单个核细胞是指外周血中使用Ficoll密度梯度离心法所得的中间云雾状的细胞层中所含的细胞,包括淋巴细胞和单核细胞以及 CD34+造血干细胞。
在另一优选例中,所述扩增后的单个核细胞主要是红系祖细胞。
在另一优选例中,所述的将神经干细胞高温培养是指在38.5-39.5度条件下培养一周至一个月,优选地,所述的神经干细胞高温培养是指在39度条件下培养一周至一个月,具体时间因仙台病毒载体是否灭活而定。
在另一优选例中,所述单个核细胞培养基其配方是基础培养基:49% (Iscove’smodified Dulbecco’s medium)IMDM,48%Ham’s F-12,1%胰岛素-转铁蛋白-硒,1%chemically defined lipid concentrate,1%L-谷氨酰胺,0.05mg/ml左旋维生素C,5mg/ml牛血清蛋白BSA,0.018ul/ml硫代甘油;加入的细胞因子和化学小分子为:100ng/ml重组人干细胞因子,10 ng/ml重组人白细胞介素3,2U/ml促红细胞生成素,40ng/ml胰岛素样生长因子IGF-1,1μM地塞米松,100μg/ml全铁转铁蛋白。
在另一优选例中,所述神经干细胞培养基配方为:基础培养基包括 DMEM/F12:Neurobasal(1:1),1×N2,1×B27,1%GlutaMAX,1%NEAA,加入的细胞因子和化学小分子包括:10ng/ml重组人白血病抑制因子(rhLIF)、 CHIR99021和SB431542。
本发明的第二方面提供了一种利用上述方法诱导神经干细胞的组合物,所述组合物包含外周血单个核细胞,仙台病毒载体,所述载体携带基因为OCT4, SOX2,c-MYC,KLF-4。
在另一优选例中,外周血中单个核细胞的提取方法为:静脉采血,使用 PBS将血液稀释后,密度梯度离心法收集单个核细胞,将离心得到的中间云雾状细胞转移至离心管,加入PBS离心,吸去上清后将细胞重悬于PBS中离心,反复3次离心,去上清后将细胞重悬于PBS中计数;优选离心时温度4℃、时间为10分钟。
在另一优选例中,所述扩增后的单个核细胞以红系祖细胞为主。
在另一优选例中,外周血中单个核细胞的扩增方法为:将离心所得单个核细胞重悬于培养基中,孵育2天后去上清更换培养基;隔天更换培养基至 14天为止;优选培养基为单个核细胞培养基,该培养基主要是促进单个核细胞中红系祖细胞的扩增,不利于淋巴细胞增殖,所得转染前的单个核细胞主要成分为红系祖细胞,孵育条件为37度,5%CO2。
在另一优选例中,仙台病毒转染方法为:将扩增后的细胞离心,计数,重悬于加入仙台病毒的单核细胞培养基中,MOI值=10,转染后置于含有培养基的12孔板中孵育;优选培养基为单个核细胞培养基,孵育条件为37度, 5%CO2。
在另一优选例中,所述的单个核细胞培养基的配方包含基础培养基和加入的化学小分子,所述基础培养基的配方为:49%(Iscove’s modified Dulbecco’s medium)IMDM,48%Ham’s F-12,1%胰岛素-转铁蛋白-硒, 1%chemically defined lipidconcentrate,1%L-谷氨酰胺,0.05mg/ml左旋维生素C,5mg/ml牛血清蛋白BSA,0.018ul/ml硫代甘油;也包含加入的细胞因子和化学小分子为:100ng/ml重组人干细胞因子,10ng/ml重组人白细胞介素3,2U/ml促红细胞生成素,40ng/ml胰岛素样生长因子IGF-1, 1μM地塞米松,100μg/ml全铁转铁蛋白。
在另一优选例中,所述的神经干细胞培养方法为利用携带基因OCT4,SOX2, c-MYC,KLF-4的仙台病毒载体转染后的细胞在单个核细胞培养基中培养2天,然后将其离心,去上清,再将细胞重悬在神经干细胞培养基中,接种在事先 Matrigel包被过夜的12孔板上,密度为3-4×105/孔,每隔2天更换一次培养基,注意观察细胞变化,在转染后10天左右,皿中会出现神经干细胞克隆,待干细胞继续扩增,在转染后20-30天左右,等到克隆足够大时,先使用 Accutase将神经干细胞消化后,使用移液枪将克隆挑出,转移到已经用多聚赖氨酸包被过夜的96孔板上,进行干细胞扩增。
上述获得的诱导神经干细胞在体外传代,扩增一月后,升高孵育温度至 39度培养一周后,进行PCR检测外源基因及载体基因,外源基因及载体基因均消失。
在另一优选例中,神经干细胞培养基也包含基础培养基和加入的小分子,基础培养基的配方为:DMEM/F12:Neurobasal(1:1),1×N2,1×B27,1% GlutaMAX,1%NEAA;加入的细胞因子和化学小分子为:10ng/ml重组人白血病抑制因子(rhLIF);CHIR99021;SB431542。
在另一优选例中,所述的从外周血中提取单个核细胞的方法包括以下步骤:
(1)室温下,成人外周静脉采血。
(2)采用Ficoll密度梯度离心法收集单个核细胞:将外周血按1:2使用PBS稀释,室温,存放在50ml离心管中,稀释后的血液若不够35ml,用PBS补齐。
(3)另取50ml离心管盛15ml Ficoll-Paque Premium,然后倾斜45度,使得稀释的血液缓慢流入到盛Ficoll-Paque Premium管中。
(4)25度,离心30分钟,离心速度为750g。
(5)离心后在离心管中上层为血浆样物,中间云雾状细胞即为我们所需要的单个核细胞层。
(6)将中间的云雾状细胞层移至另外50ml离心管中。
(7)在获得的细胞中加入30mlPBS,350g,4度,离心10分钟,此过程中要打开离心机制动。
(8)吸去上清,将细胞重悬在25ml PBS中。4度,300g,离心10分钟。
(9)吸去上清,将细胞重悬在25ml PBS中。4度,300g,离心10分钟。 (重复第8步)。
(10)去上清,将细胞重悬在5ml PBS中,计数。
在另一优选例中,所述的在单个核细胞培养基中扩增单个核细胞包括以下步骤:
(1)Day-14将离心所得的单个核细胞以2-3×106/ml重悬在单个核细胞培养基中,37度,5%CO2,2天。
(2)Day-11收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中。孵箱孵育3天。
(3)Day-8收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中。孵箱孵育4天。
(4)Day-4收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中,孵箱孵育4天。
在另一优选例中,所述的仙台病毒载体转染方法包括以下步骤:
(1)Day 0收集细胞,计数,离心,去上清,将2×106单个核细胞重悬在5mlPBS中,室温,离心5分钟,200g,去上清。
(2)将以上细胞重悬在3ml加有转染病毒的单个核细胞培养基中。
(3)孵育条件为37度,5%CO2。
在另一优选例中,所述的神经干细胞培养和扩增的方法包括:
(1)准备:在6孔板中放入1ml使用神经干细胞培养基1:100稀释的 Matrigel,孵箱过夜,备用。
(2)将转染后2天的单个核细胞离心,去上清,将细胞重悬在神经干细胞培养基中,以(2-4)×105/孔接种在6孔板上。隔天换液。在第十天左右,可见干细胞克隆出现。继续换液,使其继续扩增。
(3)第20-30天左右,克隆增大,使用移液枪将细胞吹下后转移至 PDL-Laminin包被的96孔板中,继续扩增,光镜下诱导神经干细胞具有类似神经干细胞的形态。
(4)传代扩增一月后,升高孵箱温度至39度孵育一周,然后取神经干细胞进行PCR鉴定仙台病毒载体是否灭活,如果载体已灭活可以将细胞培养箱的温度调整为37度,如果载体未灭活,继续在39度孵箱内培养传代,直至仙台病毒载体灭活。
本发明的第三方面提供了一种利用上述方法诱导的神经干细胞,所述神经干细胞能表达神经干细胞相关基因,能分化出不同种类的神经元,星形胶质细胞及少突胶质细胞。
本发明的第四方面提供了一种利用所述的神经干细胞诱导多巴胺能神经元的方法,所述的方法分为第一和第二阶段,分别使用两种不同的培养基配方,所述第一阶段培养基配方I包括基础培养基为DMEM/F12,1×N2,1×B27, 1%GlutaMAX,1%NEAA;加入的化学小分子为:SAG1和FGF8;所述的二阶段培养基配方包括基础培养基为DMEM/F12,1×N2,1×B27,1%GlutaMAX, 1%NEAA;加入的细胞因子和化学小分子为:BDNF,GDNF,AA,DAPT,cAMP,TGFβⅢ。神经干细胞体外可被定向分化成为多巴胺能神经元,且多巴胺能神经元可达到较高的比例。
在另一优选例中,所述的神经干细胞体外定向分化的多巴胺能神经元能够分泌多巴胺及其代谢产物,证明存在多巴胺能神经元的生理分泌功能。
本发明的第五方面,提供了体外诱导神经干细胞或多巴胺能前体细胞在治疗神经系统疾病中的应用,所述神经系统病变疾病为脑神经损伤、脊髓损伤、帕金森病、肌萎缩性侧索硬化症、脑卒中。优选地,所述神经干细胞或多巴胺能前体细胞在治疗帕金森疾病中的应用。
在另一优选例中,所述神经干细胞或多巴胺能前体细胞移植入脑部纹状体,8周后所见神经干细胞在体内分化,且无致瘤性。
在另一优选例中,选取所述神经干细胞诱导分化第10天到第13天的多巴胺能神经前体细胞移植到脑内纹状体区。
在另一优选例中,所述的神经干细胞分化的多巴胺能神经前体细胞能够改善帕金森病小鼠模型的行为学,明显改善小鼠帕金森病症状,证明体外分化的多巴胺能神经前体细胞在体内能够存活并代替体内损伤死亡的多巴胺能神经元分泌多巴胺,改善帕金森病症状。
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。
本发明具有以下技术优势:
1.本发明提供的诱导神经干细胞的方法,将从外周血中提取的细胞利用非整合的仙台病毒载体诱导成神经干细胞,通过升高温度后可将载体灭活,同时神经干细胞不受高温影响,能够维持神经干细胞的特性,稳定性。本发明所述的仙台病毒载体为转导入神经干细胞胞浆内的单链RNA病毒载体,不整合入神经干细胞基因组内,高温39度可以灭活,本发明的载体与整合性病毒载体及非整合性质粒载体相比,仙台病毒存在着非整合性良好,诱导过程可控性及缩短诱导时间的特点。
本发明提供的诱导神经干细胞能够在体外继续分化为多巴胺能神经元及各种神经元和胶质细胞等。另外,外周血采集方便、取材方便、创伤小,受到操作条件的影响小而不易受到污染。
2.本发明提供的诱导神经干细胞的方法,得到的神经干细胞体外扩增代数多,无致瘤性。神经干细胞全基因组测序结果提示无原癌基因、抑癌基因及神经干细胞相关基因的有害突变,说明本发明的方法安全有效。
3.本发明提供诱导神经干细胞体外定向分化为多巴胺能神经元的方法,且能够分泌多巴胺,分化方法短期,高效。
4.本发明利用多巴胺能神经前体细胞移植入帕金森病SCID小鼠模型,证明了在动物模型上细胞移植治疗帕金森病的安全性和有效性。
附图说明
图1将外周血经Ficoll密度梯度离心后出现中间云雾状的细胞层,黑色箭头所指即为云雾状单个核细胞层。
图2外周血单个核细胞在体外扩增14天后形态。
图3外周血单个核细胞体外扩增2周后经仙台病毒载体转导10天后出现神经干细胞克隆。
图4神经干细胞贴壁培养时形态,单层细胞形态类似于神经上皮细胞。
图5免疫细胞化学染色鉴定诱导神经干细胞表达神经干细胞标志Nestin 蛋白,绿色为Nestin蛋白,蓝色为DAPI细胞核。
图6免疫细胞化学染色鉴定诱导神经干细胞表达神经干细胞标志Pax6蛋白,红色为Pax6蛋白,蓝色为DAPI细胞核。
图7免疫细胞化学染色鉴定诱导神经干细胞表达神经干细胞标志Sox1蛋白,红色为Sox1蛋白,蓝色为DAPI细胞核。
图8诱导神经干细胞具有正常男性的核型,46XY。
图9免疫细胞化学染色鉴定诱导神经干细胞体外分化出成熟神经元,绿色为TUJ1蛋白,蓝色为DAPI细胞核。
图10免疫细胞化学染色鉴定诱导神经干细胞体外分化为少突胶质细胞,红色为O4蛋白,蓝色为DAPI细胞核。
图11免疫组织化学染色鉴定诱导神经干细胞可以在免疫缺陷小鼠体内分化,且无致瘤性,绿色为Nuclei蛋白,红色为NeuN蛋白,蓝色为DAPI细胞核。
图12免疫组织化学染色鉴定神经干细胞体外定向分化为多巴胺能神经元,红色为TH蛋白,蓝色为DAPI细胞核。
图13HPLC测定多巴胺能神经元生理分泌功能曲线,可以看到多巴胺能神经元能够分泌DA,DOPAC和HVA。
图14(A)PD免疫缺陷小鼠造模、细胞移植及行为学检测流程图。(B)PD 小鼠行为学检测结果,***p<0.001。细胞移植12周后可见细胞能够分化成为成熟的中脑多巴胺能神经元,TH+细胞为13.84%,FOXA2/TH的比率为 86.78%,NURR1/TH的比率为91.72%,GIRK2/TH的比率为98.77%。
图15PD小鼠细胞移植组组织学染色结果及统计图。(A)小鼠细胞移植后组织学染色结果(B)小鼠细胞移植组组织学染色统计结果。
图16诱导的神经干细胞在体外分化第10天、18天和24天不同多巴胺能神经元标志物的表达比例,分化第24天得到57%的TH阳性的多巴胺能神经元,表达A9区成熟多巴胺能神经元标记物GIRK2的比例为28%。
具体实施方式
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。
实施例1诱导外周血单个核细胞分化为神经干细胞
步骤一:分离血液中单个核细胞。
(1)室温下,成人外周静脉采血6ml,肝素抗凝管存储,上下颠倒混匀5 次。
(2)采用密度梯度离心法收集单个核细胞:将外周血按1:2使用PBS稀释,室温,存放在50ml离心管中,稀释后的血液若不够35ml,用PBS补齐。
(3)另取50ml离心管盛15ml Ficoll-Paque Premium,然后倾斜45度,使得稀释的血液缓慢流入到盛Ficoll-Paque Premium管中。
(4)25度,离心30分钟,离心速度为750g,离心时,关闭离心机制动。
(5)离心后在离心管中上层为血浆样物,中间云雾状细胞即为我们所需要的单个核细胞层,如图1所示。
(6)吸去上层,将中间的云雾状细胞层移至另外50ml离心管中。
(7)在获得的细胞中加入30mlPBS,350g,4度,离心10分钟,此过程中要打开离心机制动。
(8)吸去上清,将细胞重悬在25ml PBS中。4度,300g,离心10分钟。
(9)吸去上清,将细胞重悬在25ml PBS中。4度,300g,离心10分钟。 (重复第8步)。
(10)去上清,将细胞重悬在5ml PBS中,计数。
步骤二:扩增血液中单个核细胞。
(1)Day-14将离心所得的单个核细胞以2-3×106/ml重悬在单个核细胞培养基中,37度,5%CO2,2天。
(2)Day-11收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中。孵箱孵育3天。
(3)Day-8收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中。孵箱孵育4天。
(4)Day-4收集细胞,离心,200g,去上清,将细胞按1×106/ml重悬在单个核细胞培养基中,孵箱孵育4天。
步骤三:转染
(1)Day 0收集细胞,计数,离心,去上清,将2×106单个核细胞重悬在5mlPBS中,室温,离心5分钟,200g,去上清,如图2所示。
(2)将以上细胞重悬在3ml加有转染病毒的单个核细胞培养基中。
(3)孵育条件为37度,5%CO2。
步骤四:神经干细胞的培养
(1)准备:在6孔板中放入1ml使用神经干细胞培养基1:100稀释的 Matrigel,孵箱过夜,备用。
(2)将转染后2天的单个核细胞离心,去上清,将细胞重悬在神经干细胞培养基中,以(2-4)×105/孔接种在6孔板上。隔天换液。在第十天左右,可见干细胞克隆出现如图3所示。继续换液,使其继续扩增。
(3)第20-30天左右,克隆增大,使用移液枪将细胞吹下后转移至PDL-Laminin包被的96孔板中,继续扩增,光镜下诱导神经干细胞具有类似神经干细胞的形态如图4所示。
(4)传代扩增一月后,升高孵箱温度至39度孵育一周,然后取神经干细胞进行PCR鉴定。
实验例2诱导神经干细胞表达神经干细胞标志蛋白
将诱导神经干细胞按5×104接种在多聚赖氨酸与层黏蛋白包被的12mm玻片上,神经干细胞培养基培养48小时后进行染色,具体步骤是:
(1)吸去培养基,使用PBS洗两遍,然后加入4%多聚甲醛固定10分钟。
(2)吸去多聚甲醛,加入0.3%PBST 1ml,重复3次,每次间隔5分钟。
(3)加入3%驴血清封闭1小时,室温下。4.1小时后,加入一抗,分别将抗体如Nestin(1:500Mouse BD bioscience),Sox1(1:200Goat BD bioscience),按比例加入1%驴血清中,然后孵育细胞,4度,过夜。
(4)吸去一抗,加入对应的二抗。驴抗鼠FITC按1:200对应Nestin,驴抗山羊cy3按1:400对应Sox1,Sox2。室温,避光放置2小时。
(5)吸去二抗,PBS洗三遍,然后加入DAPI(1:1000Sigma-Aldrich),孵育10分钟。
(6)封片,激光共聚焦拍照,所见干细胞表达Nestin,Sox1,Sox2蛋白,如图5、6、7所示。将处于增殖期的诱导神经干细胞进行裂解,核型分析所示,诱导神经干细胞具有正常核型,如图8所示。
实验例3神经干细胞分化为成熟的神经元。
将神经干细胞以2×104接种在多聚赖氨酸与层黏蛋白包被的12mm玻片上,神经干细胞培养基培养24小时后,将培养基更换为神经元分化培养基,其成分为DMEM:F12,1%N2,1%B27,1%谷胺酰胺Glutamine,1%非必须氨基酸NEAA(Life Technologies),隔天换液,6周后将细胞固定,免疫细胞化学染色。具体为MAP2(1:200Mouse Sigma),Neun(1:400RabbitMillpore),表达成熟神经元蛋白Map2Neun,如图9。
实验例4神经干细胞分化为多巴胺能神经元
将神经干细胞以2×104接种在多聚赖氨酸与层黏蛋白包被的12mm玻片上,神经干细胞培养基培养24小时后,将培养基更换为神经元分化基础培养基,其成分为DMEM:F12,1%N2,1%B27,1%谷氨酰胺Glutamine,1%非必须氨基酸NEAA,第一阶段该培养基中加入化学小分子1uM SAG1(Enzo), 100ng/ml FGF8(PeproTech),培养10天后将培养基更换为第二阶段培养基,其内加入BDNF,GDNF,AA,DAPT,cAMP,TGFβⅢ。继续培养2周后进行细胞免疫化学染色。其中一抗TH(1:500Sheep Millpore)。如图10所示,诱导神经干细胞能分化出TH阳性的多巴胺能神经元。其中42.51%的TH阳性细胞同时表达A9区多巴胺能神经元markerGIRK2,说明在体外可迅速高效的得到中脑黑质特异的多巴胺能神经元。分化过程中不同时间点的阳性细胞统计如图16 所示。说明本发明的方法能够高效诱导神经干细胞分化出具有TH阳性和中脑黑质特异的多巴胺能神经元。HPLC测定多巴胺能神经元生理分泌功能如图13 所示。
实验例5神经干细胞分化为少突胶质细胞
将神经干细胞以2×104接种在多聚赖氨酸与层黏蛋白包被的12mm玻片上,神经干细胞培养基培养24小时后,将培养基更换为神经元分化基础培养基,其内加入以下小分子:1uM反维甲酸RA(Sigma-Aldrich),20ng PDGF-AB(PeproTech),10ng/ml bFGF(PeproTech),SAG1(Enzo);2周后将培养基改为神经元分化基础培养基,其内加以下小分子: 20ngPDGF-AB(PeproTech),SAG1(Enzo),60ng/ml甲状腺素 T3(Sigma-Aldrich),1mM环磷酸腺苷(Signa-Aldrich),10ng/ml胰岛素样生长因子IGF-1(PeproTech),10ng/ml神经营养因子3NT3(PeproTech),继续培养6周后,将细胞进行细胞化学染色,一抗为O1(1:300MouseeBioscience)。如图11所示,诱导神经干细胞能分化出少突胶质细胞,标志蛋白为O1,Bar等于50um。
实验例6神经干细胞体内分化并且无致瘤性。
我们选取同一母代细胞来源的两株神经干细胞进行全基因组测序,比较分析两株子代细胞与母代细胞之间的突变,发现两株子代神经干细胞均无致瘤基因的突变,无致瘤性。
将神经干细胞以1×105/μl重悬在5%葡萄糖溶液中,将免疫缺陷小鼠全麻成功后,立体定位下使用微量注射器将细胞重悬液注射到小鼠单侧纹状体。 2月后全麻下灌注小鼠,取脑组织冰冻切片染色。可见移植后的神经干细胞能在小鼠体内分化成Tuj-1阳性的细胞,如图12所示。
实验例7神经干细胞定向高效分化为多巴胺能神经前体细胞移植入PD 小鼠体内,行为学有显著性差异
将神经干细胞以5×103接种在多聚赖氨酸与层黏蛋白包被的六孔板内,神经干细胞培养基培养24小时后,将培养基更换为神经分化培养基Ⅰ,其成分为DMEM:F12,1%N2,1%B27,1%谷氨酰胺Glutamine,1%非必须氨基酸 NEAA,SAG1,FGF8,培养10天后将培养基更换为分化培养基Ⅱ,其内加入 BDNF,GDNF,cAMP,AA,TGFβⅢ,DAPT继续培养2周。用6-OHDA毁损免疫缺陷小鼠(SCID-beige)右侧纹状体形成PD模型,将2×105多巴胺能前体细胞(分化day10:day13=1:7)移植入毁损侧,之后2、4、6、8、12周进行行为学检测,发现移植细胞组PD小鼠脑内无肿瘤形成,行为学有显著性改善,与缓冲液buffer组相比均存在显著性差异(***p<0.001),PD免疫缺陷小鼠造模、细胞移植及行为学检测流程图如图14所示。组织学染色也可见到分化成熟的中脑多巴胺能神经元,结果如图15所示。
如无特别说明,本发明中所用的试剂均可从商业途径购买。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明的保护范围之中。
Claims (11)
1.一种诱导神经干细胞的方法,所述方法包含以下步骤:(1)从外周血中提取单个核细胞并在单个核细胞培养基中扩增单个核细胞;(2)利用携带基因OCT4,SOX2,c-MYC,KLF-4的仙台病毒载体转导扩增后的单个核细胞;(3)将转导的单个核细胞接种到用Matrigel包被的细胞培养板上在神经干细胞培养基中培养,继续培养直至出现神经干细胞克隆;(4)取出神经干细胞克隆,进行神经干细胞扩增,期间将神经干细胞高温培养,获得仙台病毒被灭活的神经干细胞。
2.如权利要求1所述的诱导神经干细胞的方法,其特征在于,所述单个核细胞为外周血中使用Ficoll密度梯度离心法所得的中间云雾状的细胞层中所含的细胞,包括淋巴细胞和单核细胞以及CD34+造血干细胞。
3.如权利要求1-2任一项所述的诱导神经干细胞的方法,其特征在于,所述扩增后的单个核细胞主要是红系祖细胞。
4.根据权利要求1-3任一项所述的诱导神经干细胞的方法,其特征在于,所述将神经干细胞高温培养是在38.5-39.5度条件下培养一周至一个月,优选在39度条件下直至仙台病毒被灭活。
5.根据权利要求1-4任一项所述的诱导神经干细胞的方法,其特征在于,所述单个核细胞培养基,其配方是基础培养基:49%(Iscove’s modified Dulbecco’s medium)IMDM,48%Ham’s F-12,1%胰岛素-转铁蛋白-硒,1%chemically defined lipid concentrate,1%L-谷氨酰胺,0.05mg/ml左旋维生素C,5mg/ml牛血清蛋白BSA,0.018ul/ml硫代甘油;加入的细胞因子和化学小分子为:100ng/ml重组人干细胞因子,10ng/ml重组人白细胞介素3,2U/ml促红细胞生成素,40ng/ml胰岛素样生长因子IGF-1,1μM地塞米松,100μg/ml全铁转铁蛋白。
6.根据权利要求1-5任一项所述的诱导神经干细胞的方法,其特征在于,所述神经干细胞培养基配方为:基础培养基包括DMEM/F12:Neurobasal(1:1),1×N2,1×B27,1%GlutaMAX,1%NEAA,加入的细胞因子和化学小分子包括:10ng/ml重组人白血病抑制因子(rhLIF)、CHIR99021和SB431542。
7.在权利要求1-6任一项所述方法中使用的一种用于诱导神经干细胞的组合物,所述组合物包含外周血单个核细胞,仙台病毒载体,所述载体携带基因为OCT4,SOX2,c-MYC,KLF-4。
8.一种神经干细胞,其特征在于,根据权利要求1-7任一项所述的诱导方法获得,所述神经干细胞能表达神经干细胞相关基因,能分化出不同种类的神经元,包括多巴胺能神经元等,以及不同种类的胶质细胞。
9.一种诱导神经干细胞产生多巴胺能神经前体细胞的方法,其特征在于,根据权利要求1-5任一项所述方法得到的神经干细胞,所述诱导过程分为第一和第二阶段,分别使用两种不同的培养基配方,所述第一阶段培养基配方I包括基础培养基为DMEM/F12,1×N2,1×B27,1%GlutaMAX,1%NEAA;加入的化学小分子为:SAG1和FGF8,培养10天后将培养基更换为第二阶段培养基配方Ⅱ;所述的二阶段培养基配方包括基础培养基Ⅱ为DMEM/F12,1×N2,1×B27,1%GlutaMAX,1%NEAA;加入细胞因子和的化学小分子为:BDNF,GDNF,AA,DAPT,cAMP,TGFβⅢ,继续培养2周。
10.根据权利要求8所述的神经干细胞或权利要求9所述的方法产生的多巴胺能神经前体细胞在治疗脑神经损伤、脑卒中、脊髓损伤、帕金森病、肌萎缩性侧索硬化症中的应用,优选地是在治疗帕金森病中的应用。
11.根据权利要求10所述的多巴胺能神经前体细胞在治疗帕金森病中的应用,其特征在于,使用如权利要求9所述方法,选取神经干细胞诱导分化第10天到第13天的多巴胺能神经前体细胞移植到脑内纹状体区。
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