CN111334473A - 一种人成体神经干细胞的制备方法及其防治脑卒中的用途 - Google Patents
一种人成体神经干细胞的制备方法及其防治脑卒中的用途 Download PDFInfo
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Abstract
本发明公开了一种人成体神经干细胞的制备方法及其防治脑卒中的用途。具体而言,此种方法建立的细胞系高表达神经干细胞标记物Nestin、Musashi、Sox2、CD133等,传代至P20代时依然保持较高的干性,稳定性高。此细胞系培养上清对过氧化氢、氧糖剥夺所致神经细胞损伤具有显著改善作用,对脑卒中急性期及后遗症期动物模型均具有显著改善作用,可以作为脑卒中防治药物应用。
Description
技术领域
本发明属于细胞生物学、神经药理学领域,具体涉及一种人成体神经干细胞的制备方法及其防治脑卒中的用途。
背景技术
我国已进入老龄化社会,老龄化速度已快于总人口增长速度。各种老年病患者人数和比例也呈上升趋势。脑卒中作为中老年人的多见病,在我国已经成为了仅次于心血管病的第二大死亡杀手,严重威胁人类生命健康。目前尚未有防治此类疾病的特效药物,因此开发出具有靶向性、副作用小、能有效控制病情的药物势在必行。
神经干细胞(neural stem cells,NSCs)是一种具有自我更新能力和多向分化潜能的细胞。近年来,随着干细胞技术的研究和发展,研究人员发现神经系统损伤疾病发生时损伤丢失的神经细胞可以通过移植干细胞来修复,且这种方法十分有效。
但是,常规NSCs培养方法最终能获得的细胞数量有限,不能满足临床治疗需要。一方面,要达到对多人的治疗效果,通常需要获得多个胚胎组织样本,如此存在较大的伦理学争议。另一方面,不同样本来源的细胞生物学性状存在差异,由此导致的结果也存在一定的变异性,因而影响了科学数据的准确性。因此,建立稳定的可长期扩增的 NSCs细胞系,此细胞系在传代20代以上时,依然保持NSCs生物学性状不变,可以有效的进行临床研究。
发明内容
鉴于以上问题,本发明的主要目的在于提供一种人成体神经干细胞系的建立方法及其在脑卒中防治中的用途。
本发明提供的该种神经干细胞的传代方法,包括神经干细胞培养基和神经干细胞传代培养两个步骤。
所述神经干细胞传代培养基由基础培养基、B27、表皮生长因子、碱性成纤维细胞生长因子、胰岛素、腐胺、Notch 1、WNT3a、黄体酮、亚硒酸钠、胰岛素生长因子、层粘蛋白组成;B27为1×,表皮生长因子浓度20ng/ml,成纤维细胞生长因子浓度20 ng/ml,胰岛素10mg/ml,腐胺16.2mg/L,Notch 150nmol/L,WNT3a20nmol/L,黄体酮20nmol/L,亚硒酸钠5ug/L,胰岛素生长因子25mg/ml,层粘蛋白5μg/ml,所述基础培养基由DMEM/F12、HEPES和碳酸氢钠组成,其中,DMEM/F12为1×,HEPES 浓度为20mmol/L,碳酸氢钠浓度为10mmol/L。
其中,所述神经干细胞传代培养具体包括以下步骤:
1).半量换液及收集神经干细胞条件培养基:当神经干细胞原代培养的半数神经球直径超过150μm时,竖立细胞培养瓶2分钟,使神经干细胞球沉降至瓶底,吸出半量培养基之后,加入等量的新鲜神经干细胞原代培养基,换液完毕后放回细胞培养箱继续培养。再将上述吸出的半量培养基收集到离心管中,500g/min离心5分钟,收集上清即可获得神经干细胞条件培养基,保存4℃备用;
2).预处理:在神经干细胞传代前一天,向新的T75细胞培养瓶中加入5ml神经干细胞条件培养基,备用;
3).消化传代:当神经干细胞培养7-9天约半数神经球直径超过250μm时,将神经干细胞及培养基收集到50ml离心管中,500g/min离心5分钟,收集上清即可获得神经干细胞条件培养基,保存4℃备用。将神经干细胞球重悬于10倍体积Accutase消化液中,置于37℃培养箱消化10分钟,待可见细胞球略微松散时,加入胰酶抑制剂终止消化,采用巴斯德吸管将细胞轻轻吹打分散;500g/min,5分钟,离心收集细胞;
4).培养:取出前一天采用神经干细胞条件培养基预处理的细胞培养瓶,吸弃其中的培养基;将神经干细胞传代培养基与神经干细胞条件培养基1∶1混合,之后采用此混合培养基重悬消化后的神经干细胞,并按1∶3传代接种于预处理过的细胞培养瓶;将传代后的神经干细胞置于37℃细胞培养箱中培养;
5).换液:传代3-4天后,进行半量换液:竖立细胞培养瓶2分钟,使神经干细胞球沉降至瓶底,离心管吸出半量培养基之后,加入等量的新鲜神经干细胞传代培养基,换液完毕后放回细胞培养箱继续培养,再将上述吸出的半量培养基收集到离心管中, 500g/min离心5分钟,收集上清即可获得神经干细胞条件培养基,保存4℃备用;
6).重复按步骤2)-5)方法进行神经干细胞的传代并培养,建立稳定的神经干细胞系。
上述神经干细胞系的建立方法,最后还包括有细胞冻存步骤,所述细胞冻存步骤具体为:
细胞传代后7-9天进行细胞冻存,将神经干细胞收集至离心管中,500g离心5分钟,收集细胞;将收集到的细胞重悬于细胞冻存液(DMEM/F12培养基:人白蛋白:二甲基亚砜=7:2:1)中,冻存密度5×106/ml;将冻存细胞置于异丙醇冻存盒中,-80℃过夜;将细胞转移至液氮罐中长期保存。
在建系过程中,观察干细胞标志物及干细胞分化标记的表达。所获得的神经干细胞系呈球状悬浮生长,可以长期稳定增殖超过20代,细胞倍增时间约为3-4天,高表达 Nestin、Musashi、CD133、Sox2等神经干细胞标志物。
通过本发明所述的一种神经干细胞系的建立方法所建立的神经干细胞系,应用于防治脑卒中。
本发明具有以下有益效果:
神经干细胞短期培养相对容易,但建系长期培养却很困难,究其原因是在长时间培养过程中容易产生细胞分化,丧失干细胞特性。本发明可以建立稳定的神经干细胞系,进行长期培养获得大量足够研究需求的细胞,同时还能保证细胞的稳定性,尽可能减少研究误差。它在GMP控制条件下进行分离并长期培养,不接触任何动物源性成分,没有致瘤性,可高效表达Nestin、Musashi、CD133、Sox2等神经干细胞标志物。
采用条件培养基预处理培养瓶,尽可能降低培养瓶带来的细胞分化问题。如果不采用条件培养基预处理,传代后的神经干细胞容易发生贴壁从而分化的现象,预处理后几乎很少细胞贴壁。
神经干细胞系建立在GMP控制下进行,且在培养、冻存过程中不使用任何抗生素及动物源性成分,对细胞未来可能的临床试验研究的安全性保证做到了最大。
附图说明
图1倒置显微镜观察不同代次神经干细胞球形态;
图2神经干细胞干性标记Nestin、Musashi、Sox2、CD133、CD29及分化标记Tuj- 1、GFAP、O4流式鉴定图;
图3神经干细胞干性标记Nestin、Musashi、CD133及分化标记Tuj-1、GFAP、O4 免疫荧光图;
图4脑缺血再灌注2h、48h各组动物Longa’s评分情况。其中,干细胞治疗组动物能显著降低Longa’s评分分数,改善动物行为学。means±SEM,n=15,**p<0.01 vs 48h模型对照组;
图5TTC染色检测脑缺血再灌注及干细胞治疗48h各组动物脑梗死情况。means± SEM,n=15,***p<0.001vs模型对照组;
图6脑缺血再灌注28天各组动物脑萎缩情况。means±SEM,n=13-15,*p<0.05 vs模型对照组;
图7脑缺血再灌注28天各组动物脑皮层中段及纹状体病理情况。A,皮层中段;B,纹状体。
具体实施方式
以下结合附图及实施例对本发明的技术方案进一步说明,但不作对其的限定:
实施例1:神经干细胞的原代培养、传代培养和冻存
本实施例中所用各种液体说明如下:
1、基础培养基:由DMEM/F12、HEPES和碳酸氢钠组成。其中,DMEM/F12为1 ×;HEPES为20mmol/L;碳酸氢钠为10mmol/L。
2、神经干细胞原代培养基:由基础培养基、B27、表皮生长因子、碱性成纤维细胞生长因子、胰岛素、腐胺、Notch 1、WNT3a、黄体酮、亚硒酸钠、胰岛素生长因子、层粘蛋白组成;B27为1×,表皮生长因子浓度20ng/ml,成纤维细胞生长因子浓度 20ng/ml,胰岛素10mg/ml,腐胺16.2mg/L,Notch 150nmol/L,WNT3a20nmol/L,黄体酮20nmol/L,亚硒酸钠5ug/L,胰岛素生长因子25mg/ml,层粘蛋白5μg/ml,所述基础培养基由DMEM/F12、HEPES和碳酸氢钠组成。
3、神经干细胞传代培养基:由基础培养基、表皮生长因子、碱性成纤维细胞生长因子、胰岛素、腐胺、Notch 1、WNT3a、黄体酮、亚硒酸钠、胰岛素生长因子、层粘蛋白组成;表皮生长因子浓度20ng/ml,成纤维细胞生长因子浓度20ng/ml,胰岛素10 mg/ml,腐胺16.2mg/L,Notch 150nmol/L,WNT3a 20nmol/L,黄体酮20nmol/L,亚硒酸钠5ug/L,胰岛素生长因子25mg/ml,层粘蛋白5μg/ml,所述基础培养基由 DMEM/F12、HEPES和碳酸氢钠组成。
4、神经干细胞冻存液:由基础培养基、人白蛋白和二甲基亚砜组成。其中, DMEM/F12培养基:人白蛋白:二甲基亚砜=7:2:1。
5、神经干细胞消化液:Accutase每107个细胞加入10ml。
本实施例包括以下步骤:
1.当神经干细胞原代培养3-4天、半数神经球直径超过150μm时,半量换液(竖立细胞培养瓶2分钟,使神经干细胞球沉降至瓶底,吸出半量培养基之后,加入等量的新鲜神经干细胞原代培养基),换液完毕后放回细胞培养箱继续培养。再将上述吸出的半量培养基收集到离心管中,500g/min离心5分钟,收集上清即可获得神经干细胞条件培养基,保存4℃备用;
3.在神经干细胞传代前一天,向新的细胞培养瓶中加入10ml神经干细胞条件培养基,备用。
4.当神经干细胞培养7-9天约半数神经球直径超过250μm时,将神经干细胞及培养基收集到50ml离心管中,500g/min离心5分钟,收集上清即可获得神经干细胞条件培养基,保存4℃备用。将神经干细胞球重悬于Accutase消化液中,置于37℃培养箱消化5分钟,待可见细胞球略微松散时,加入胰酶抑制剂终止消化,采用巴斯德吸管将细胞轻轻吹打分散;500g/min,5分钟,离心收集细胞;
5.取出前一天采用神经干细胞条件培养基预处理的细胞培养瓶,吸弃其中的培养基;将神经干细胞传代培养基与神经干细胞条件培养基1∶1混合,之后采用此混合培养基重悬消化后的神经干细胞,并按1∶3传代接种于预处理过的细胞培养瓶;将传代后的神经干细胞置于细胞培养箱中培养。
6.此后按步骤3-5方法进行神经干细胞的培养,并定期进行神经干细胞的鉴定。如此可建立稳定的神经干细胞系,获得足量的神经干细胞备科研或临床试验研究。
7.神经干细胞冻存:神经干细胞传代后3-4天可进行细胞冻存,将神经干细胞收集至离心管中,500g/min离心5分钟,收集细胞。将收集到的神经干细胞重悬于细胞冻存液中,冻存密度5×106/ml。冻存液成分为DMEM/F12培养基:人白蛋白:二甲基亚砜= 7:2:1体积比。
8.不同代次神经干细胞球培养7-9天时,形态如图1所示。呈现球状悬浮状体,约250μm左右,球体周围有毛刺。
实施例2:神经干细胞的干性鉴定——流式细胞术
方法及结果:选取不同代数培养7-9天神经干细胞球,将其消化为单细胞,采用流式细胞术,对其主要干性标记Nestin、Musashi、CD133、Sox2进行测定。表面标记 CD133具体为:离心收集每管细胞至少106个,500g离心5min,弃上清,用4℃预冷 PBS洗细胞两次。加入200μL PBS含相应的抗体。涡旋3s,4℃避光孵育40-50min。直接向离心管中加入2mL PBS,4℃4000g离心6min,弃上清,重复一次。加入350-500 μL PBS重悬细胞,进行流式操作即可。其它胞内标记具体为:在加入抗体前需要经过破膜处理,其它与表面标记一致。结果如所示,Nestin、Musashi、Sox2、CD133、 CD29的阳性率分别为96%、97%、67%、91%和99%,说明此神经干细胞保持着较强的干性;神经元和星形胶质细胞分化标记Tuj-1和GFAP分别为40%和43%,少突胶质细胞分化标记为0%,说明神经干细胞球有向神经元和星形胶质细胞分化的趋势。
实施例3:神经干细胞的干性鉴定——免疫荧光
方法及结果:选取培养7-9天神经干细胞球,将细胞球做5μm冰冻切片,并对其进行干性标记Nestin、Musashi、CD133及分化标记Tuj-1、GFAP、O4免疫荧光染色。具体为,冰冻切片室温晾干15min,用组化笔将待测细胞球群圈好,置PBS中浸泡10min 以去除OCT;用含10%羊血清的PBS室温封闭切片1h,去除封闭液,加入单抗(1: 200),4℃过夜;PBS洗3次,每次5min;加入相应荧光二抗(1:100),室温孵育 1h;PBS洗3次,每次10min;甘油封片,荧光显微镜下拍照。结果如图3所示, Nestin、Musashi、CD133干性标记的阳性率较高,约大于80%,分化标记Tuj-1、GFAP 约为50%,分化标记O4几无表达。说明此干细胞干性较强,主要分化为神经元和星形胶质细胞。
实施例4:神经干细胞的裸鼠致瘤性实验
方法及结果:观察皮下接种传代后培养7-9天的神经干细胞对NOD/SCID小鼠肿瘤发生率的影响,评价其致瘤性。实验分为空白组(同体积生理盐水)、阴性对照组(人视网膜色素上皮细胞2×107)、阳性对照组(HepG2细胞2×107)、神经干细胞2×107 5组,分别将肝癌细胞HepG2、人视网膜色素上皮细胞、传代至第10代的神经干细胞接种于裸鼠背部皮下,于注射后12周进行解剖检查,观察裸鼠移植部位肿瘤形成情况。结果除阳性对照组,其它各组均无肿块形成,说明神经干细胞无致瘤性。
实施例5:干细胞培养上清对过氧化氢损伤的原代神经元保护作用
方法:取新生大鼠脑皮层细胞,用生长培养基调整细胞浓度为1×106/mL,接种至预先用0.1mg/mL多聚赖氨酸包被(18-24h)过的96孔培养板,置37℃,5%CO2孵箱培养,48小时后全量换液以去除悬浮的死亡细胞。第4-5天加阿糖胞苷(终浓度10 μmol/L)处理细胞24小时,以抑制胶质细胞生长。以后每3-4天半量换液,6-9天开始实验。取原代神经元细胞,弃去原培养液,加入含10%FBS和10%ES的DMEM/F12完全培养液,用吸管轻轻吹打使细胞分散完全,以2×106个/ml密度,每孔100μl接种于预先用多聚赖氨酸(0.1mg/ml)处理过的96孔培养板中,培养24h即可用于实验。实验分为空白组、模型组和加药组。空白组给予完全培养基,过氧化氢(H2O2)造模组是加入终浓度为100μM H2O2,加药组在造模的同时不同配比换入干细胞培养上清。5%CO2培养箱孵育 24h后,加入10μl 5mg/ml MTT,4h后形成甲臜结晶,加入100μl三联液溶解结晶,酶标仪570nm波长测定吸光度值,表示细胞存活率。
干细胞培养上清对过氧化氢损伤的原代神经元保护作用结果。如表1,过氧化氢损伤可明显减少原代神经元存活率,25%、50%换液干细胞培养上清对该损伤有显著的改善作用。
表1干细胞培养上清对过氧化氢损伤的原代神经元保护作用(means±SD,n=6)
###p<0.001vs空白对照组,**p<0.01,***p<0.001vs模型对照组
实施例6:干细胞培养上清对氧糖剥夺/复氧损伤的原代神经元保护作用
方法:神经元培养及处理方法参见实施例5。实验分为空白组、模型组和加药组。空白组给予完全培养基,氧糖剥夺/复氧组为加入低糖DMEM配置的2mM连二亚硫酸钠 (Na2S2O4)孵育2h后,再加入完全培养基复氧24h,加药组在造模的同时不同配比换入干细胞培养上清。然后加入10μl 5mg/ml MTT,4h后形成甲臜结晶,加入100μl三联液溶解结晶,酶标仪570nm波长测定吸光度值,表示细胞存活率。
干细胞培养上清对氧糖剥夺/复氧损伤的原代神经元保护作用筛选结果见表2。如表2,氧糖剥夺/复氧可明显减少原代神经元存活率,25%、50%换液干细胞培养上清对氧糖剥夺/复氧导致的原代神经元损伤均有很好的改善作用。
表2干细胞培养上清对氧糖剥夺/复氧损伤的原代神经元保护作用(means±SD, n=6)
###p<0.001vs空白对照组,**p<0.01,***p<0.001vs模型对照组
实施例7:神经干细胞改善缺血性脑卒中急性期模型大鼠脑损伤
实验设计及分组给药:SD大鼠,雄性,平均体重270-290g,维通利华 (SCXK(京)2016-0011),做单侧大脑中动脉闭塞脑缺血再灌注模型。脑缺血再灌注模型成功的标志为动物缺血再灌注损伤清醒后Zea Longa评分为1-3分。将不符合标准的动物剔出实验,其它动物进入后续实验。实验动物分为MCAO/R模型组、MCAO/R+神经干细胞脑室注射(2×106个)组两组,每组15只。大鼠脑缺血120min后再灌注,再灌注同时模型组及给药组分别右侧脑室注射生理盐水和神经干细胞2×106个,24h后采用神经功能缺失体征评分评价行为学,48h后采用TTC染色评价脑梗死情况。
大鼠脑缺血再灌注模型的建立:1、大鼠术前12h禁食不禁水,大鼠经麻醉(4%水合氯醛、1ml/100g)后用纸绷带固定大鼠四肢使其仰卧于手术台上,剔除大鼠颈部被毛,并用75%酒精消毒。2、事先准备好4根6cm的5-0缝合线,颈部正中央纵形剪开皮肤3cm;钝性分离皮下肌肉,分离到气管前肌后,沿右侧胸锁乳突肌腱向下分离,见到颈动脉鞘后上拉钩暴露颈总动脉。3、分离出颈总、颈外、颈内动脉,结扎颈总(蛙心夹结扎)、颈外动脉远心端(死结)、颈内动脉(活结),颈外动脉近心端挂线(白色线)。4、用眼科剪将颈外动脉剪一小口,插入线栓,当线栓成功插入颈外动脉时,将颈外动脉近心段挂的白线结扎(死结),剪断颈外动脉。5、松开颈内动脉活结,继续插入线栓至大脑中动脉起始端(鱼线标记处),取下蛙心夹剪掉线头。6、缺血2小时后,拔出线栓(注:缺血过程中动物有苏醒迹象要及时补麻药,剂量为初始剂量的20%)。7、松开颈总动脉蛙心夹,滴加少量青霉素以防止感染,缝合皮下肌肉和皮肤,并在对皮肤进行消毒后放入鼠笼中。8、将大鼠放入鼠笼后使用注射器向其口中滴加少量清水,苏醒 2h后根据神经功能缺失体征评分选取评分在1-3分的大鼠进行后续实验。
神经功能缺失体征评分及实验结果:参考Longa及Bederson的5分制法在动物麻醉清醒后进行评分。0分:无神经损伤症状;1分:不能完全伸展对侧前爪;2分:向对侧转圈;3分: 向对侧倾倒;4分:不能自发行走,意识丧失。分值越高,说明动物行为障碍越严重。如图4,脑缺血2h两组动物行为学无差异,在给予干细胞治疗48h后,治疗组大鼠行为学评分显著低于模型组,说明干细胞脑室注射可以显著改善大鼠运动行为学障碍。
TTC染色法测定脑梗死体积:干细胞治疗48h后,大鼠经水合氯醛(0.35 ml/100g)麻醉,断头取脑并用切片槽切成6片2mm厚的冠状切片。然后迅速将脑片置 1.5%TTC水溶液中,避光,37℃孵育15min,期间每隔5min摇动一次。经染色后,正常脑组织呈玫瑰红色,而梗死组织呈白色,且界限分明。温孵完毕后,将脑片放入装有 4%多聚甲醛的离心管中保存,次日将每组脑片排列整齐,用数码相机将脑片拍摄保存。大鼠脑梗死体积计算公式=梗死体积/未梗死侧半脑体积×100%。由图5可见,模型组有大面积灰色脑梗死,给予干细胞治疗可显著降低梗死体积。
实施例8:神经干细胞改善缺血性脑卒中后遗症模型大鼠脑损伤
实验设计及分组给药:健康雄性SD大鼠,体重270-290g,做单侧大脑中动脉闭塞脑缺血再灌注模型。脑缺血再灌注模型成功的标志为神经功能缺失体征评分为1-3。将不符合标准的动物剔出实验,其它动物进入后续实验。实验动物分为空白组、 MCAO/R模型组、MCAO/R+神经干细胞脑室注射(NSCs,5×106个)组三组,每组13- 15只。大鼠脑缺血120min后再灌注,再灌注后28天,模型组及给药组分别右侧脑室注射生理盐水和神经干细胞球,给药28天后PBS及多聚甲醛心脏灌注取脑,用于病理检测。空白组不做任何处理。
大鼠脑缺血再灌注模型的建立参考实施例7。
脑萎缩情况:大鼠经水合氯醛(0.35ml/100g)麻醉,心脏灌注取脑,大鼠脑萎缩面积计算公式=(未萎缩侧面积-萎缩侧面积)/未萎缩侧脑面积×100%。由图6可知,脑缺血再灌注28天后,大鼠缺血侧脑组织出现大面积脑萎缩现象,给予干细胞治疗可显著降低脑萎缩面积。
病理情况:将上述大鼠大脑脱水、包埋,并选取脑皮层中段、纹状体做石蜡切片,采用Nissl染色检测脑组织损伤情况。由图7可知,空白组大鼠脑皮层及纹状体细胞胞体较饱满、胞核清晰透亮、可见核仁,模型组皮层及纹状体可见大面积萎缩、丢失情况,剩余组织多见胞体萎缩浓染现象。干细胞治疗组虽然也可见浓染现象,但是其显著降低了大鼠脑皮层及纹状体萎缩情况,可明显改善其病理损伤。
Claims (5)
1.一种人成体神经干细胞系的建立,其中包括神经干细胞的培养方法和传代方法两个步骤:
1).神经干细胞条件培养基来源:当神经干细胞培养3-4天约半数神经球直径超过150μm或培养7-9天约半数神经球直径超过250μm时,吸出半量培养基,加入等量的新鲜培养基,放回细胞培养箱继续培养。将上述吸出的培养基,500G/min离心5分钟,上清即为神经干细胞条件培养基,保存4℃备用;
2).细胞培养瓶预处理:取适量神经干细胞条件培养基孵育新的细胞培养瓶8h,备用;
3).神经干细胞球的消化与传代:培养7-9天、半数神经球直径超过250μm时,500g/min离心5分钟,再将神经干细胞球重悬于Accutase消化液中,置于37℃水浴消化5分钟,超净台机械吹打15-20下,水浴消化3min,加入胰酶抑制剂终止消化,继续吹打15-20下,可见细胞球变小并多为单细胞,500g/min离心5分钟。取出神经干细胞条件培养基预处理过的细胞培养瓶,吸弃其中的培养基,将神经干细胞基础培养基与条件培养基1∶1混合,之后采用此混合培养基重悬消化后的神经干细胞,并按1∶3传代接种于预处理过的细胞培养瓶,并于CO2细胞培养箱中培养。传代3-4天后,进行半量换液。
4).重复此方法进行神经干细胞的传代和培养,建立稳定的神经干细胞系。
2.根据权利要求1-2任意一项所述的一种神经干细胞系的建立方法,其特征在于,所述神经干细胞培养基由基础培养基、B27、表皮生长因子、碱性成纤维细胞生长因子、胰岛素、腐胺、Notch 1、WNT3a、黄体酮、亚硒酸钠、胰岛素生长因子、层粘蛋白组成;B27为1×,表皮生长因子浓度15-25ng/ml,碱性成纤维细胞生长因子浓度15-25ng/ml,胰岛素5-15mg/ml,腐胺12-18mg/L,Notch 140-60nmol/L,WNT3a 10-30nmol/L,黄体酮10-30nmol/L,亚硒酸钠2-7ug/L,胰岛素生长因子20-30mg/ml,层粘蛋白2-8μg/ml,所述基础培养基由DMEM/F12、HEPES和碳酸氢钠组成,其中,HEPES浓度为15-25mmol/L,碳酸氢钠浓度为15-20mmol/L。
3.根据权利要求1-3任意一项所述的一种人神经干细胞系的建立方法,其特征在于,其包括细胞冻存步骤。所述细胞冻存步骤为:
细胞传代后7-9天进行细胞冻存,将神经干细胞收集至离心管中,500g离心5分钟,收集细胞;将收集到的细胞重悬于细胞冻存液(DMEM/F12培养基:人白蛋白:二甲基亚砜=7:2:1)中,冻存密度5×106/ml;将冻存细胞置于异丙醇冻存盒中,-80℃过夜;将细胞转移至液氮罐中长期保存。
4.通过权利要求1-3任意一项所述的一种人神经干细胞系的建立方法所建立的人神经干细胞系作为治疗脑卒中药物中的应用。
5.通过权利要求1-3任意一项所述的一种人神经干细胞系的建立方法所建立的人神经干细胞系分化所得的神经元、星形胶质细胞和少突胶质细胞作为治疗脑卒中药物中的应用。
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CN105219729A (zh) * | 2015-09-28 | 2016-01-06 | 首都医科大学宣武医院 | 一种利用非整合质粒载体诱导神经干细胞的方法及其用途 |
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