CN116445410A - 一种调控神经干细胞分化的方法及其所用的生物制品 - Google Patents
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Abstract
本发明提供了一种调控神经干细胞分化的方法,所述调控方法是指通过激活信号通路或抑制分化抑制因子的方式,诱导神经干细胞的分化;并提供了其所使用的生物制品。本发明的的特点及优点是:本发明过表达ALKBH5可促进神经干细胞分化为神经元和星形胶质细胞,ALKBH5可通过抑制分化抑制因子ID3的表达,促进神经干细胞分化。选择C17.2细胞系作为神经干细胞分化的种子细胞,不需要制备,可以直接进行诱导分化,分化周期仅为4天,所需添加的试剂仅有2‑3种,且为一次性加入,分化诱导期间不需要更换培养基,分化成功鉴定标志为NeuN(分化为神经元)和GFAP(分化为星形胶质细胞),鉴定过程简单明了,实施成本较低。
Description
技术领域
本发明涉及干细胞分化技术领域,具体涉及一种调控神经干细胞分化的方法及其所用的生物制品。
背景技术
干细胞疗法已被提议作为神经退行性疾病的治疗选择,但最佳的干细胞来源和神经再生的治疗效果仍不确定。成年哺乳动物神经干细胞是一类具有分化能力、自我更新能力等特性的细胞,它们存在于特定的脑区中,即侧脑室的室管膜下区(SVZ)及海马齿状回(DG)等区域。神经干细胞有多种来源,如人胚胎干细胞,人胎脑源性神经干细胞,人诱导多能干细胞,直接重编程星形胶质细胞等。干细胞科学对于研究和治疗都是很有前途的工具。诱导多能干细胞似乎对人类神经元研究非常有用,可以创造特定的神经元群体,特别是对于神经发育和神经退行性疾病以及缺血性损伤。神经干细胞科学在干细胞治疗和研究方面有着广阔的前景。然而,仍然需要进一步的研究来克服障碍。中枢神经***的再生一直是困扰医学界的难题之一,而经干细胞尤其是成年哺乳动物脑组织中神经干细胞的发现具有极其重大的意义,成为20世纪后10余年神经生物学领域最为重要的进展之一。它具有非常广阔的生物学研究和临床应用前景,已成为中枢神经***损伤和疾病研究的焦点。
神经干细胞:一类可以分化为神经元、星形胶质细胞和少突胶质细胞的细胞。神经干细胞的增殖、迁移、分化机制的阐明对神经干细胞研究是十分重要的。NSCs有两种细胞***方式,即不对称***和对称***。在不对称***过程中,特定的细胞***被激活,导致控制细胞质***的蛋白质失衡,并被分解以产生新的干细胞和祖细胞。通过使用这种***使得神经细胞多样化。而对称***产生两个干细胞或两个祖细胞。NSCs的增殖和分化受多种因素的影响,包括微环境、遗传调控、细胞因子、神经营养因子等因素。其中,生长因子在神经干细胞增殖分化中的作用受到了进一步的关注,如下表1所示。
表1生长因子在神经干细胞增殖分化中的作用
目前诱导神经干细胞分化的技术主要包括骨髓干细胞诱导分化技术和程序性诱导分化技术。
骨髓干细胞诱导分化技术见于郑宏志等的《诱发神经分化的方法及组合物》。其主要过程是以神经营养因子及/或二丁基环磷酸腺苷(db-cAMP)处理骨髓干细胞,其中,该神经营养因子包含衍生自神经胶细胞株的神经营养因子(GDNF)或垂体腺苷酸环化酶激活肽(PACAP),GDNF的含量为20-50ng/mL,二丁基环磷酸腺苷(db-cAMP)的含量为100μM,骨髓干细胞源自人类骨髓的尺寸筛选干细胞以3μm孔筛筛选。神经分化标志包括神经纤维轻蛋白质(NF-L)增加、α-微管蛋白(α-tubulin)增加,液泡蛋白质-突触素-1(synapsin-1)产生,神经祖细胞标记物-中间粘连蛋白(internexin)产生,细胞突起延长,以及突起分支增加。
程序性诱导分化技术见于郭阳等的《一种提高神经干细胞诱导效率和促进神经元分化的方法》。其主要过程是,(1)SMSINS细胞的分化诱导:第1天和第2天用LDN193189(Selleck S2618,0.25μM)、SB431542(Selleck S1067,1μM)处理小鼠胚胎成纤维细胞MEFs;第3天和第4天使用CHIR99021(Selleck S2924,3μM)和VPA(SigmaP4543,0.5μM);第5天和第6天使用CHIR99021(Selleck S2924,3μM)、DAPT(Selleck S2215,5μM);在第7天和第8天改善神经分化添加Shh(PeProtech 315225,100ng/ml)和Purmorphamine(selleck S3042,1μM);在第9天和第10天移除所有的小分子化合物,换NSC全培养基;此时SMSINS细胞可被胰蛋白酶化为单细胞,可在NSC全培养中接种到未涂覆PDL/L的24孔板中。(2)SMSINS细胞的体外神经元分化:将SMSINS接种于涂覆PDL/L的N2-B27培养基中[50%Neurobasal Plus(Gibco)和50%DMEM/F12培养基中,添加1×N2 supplement,1×B27 supplement,1×GlutaMax],补充10ng/ml BDNF(Peprotech)和200μM抗坏血酸(Sigma)中培养至少2周;进一步相对成熟的神经元诱导分化,则需要加入100ng/ml Shh和1μM维甲酸(Sigma)培养4天,之后切换为N2-B27培养基添加10ng/ml BDNF,10ng/ml GDNF(Peprotech),2μM cAMP(Sigma)和200μM抗坏血酸培养4周。
骨髓干细胞诱导分化技术和程序性诱导分化技术均存在一定的缺点。
骨髓干细胞诱导分化技术的缺点:
(1)种子细胞需要制备:种子细胞为骨髓干细胞,需要取骨髓,为有创操作,并需要在有资质的单位开展;获得的骨髓需要经3μm孔筛筛选而得。
(2)分化成功验证较为复杂:利用该技术验证是否分化成功,需要检测经纤维轻蛋白质(NF-L)、α-微管蛋白(α-tubulin)、液泡蛋白质-突触素-1(synapsin-1)、神经祖细胞标记物-中间粘连蛋白(internexin)、细胞突起、以及突起分支等几类指标。
这些缺点增加了利用该技术诱导神经干细胞分化的材料成本、人工成本、受试者健康风险、操作复杂度和制备周期。
程序性诱导分化技术的缺点是分化周期长、操作步骤多。SMSINS细胞的分化诱导阶段需要10天,需要添加8类化学试剂和更换1次培养基,并且需要二次接种;SMSINS细胞的体外神经元分化阶段至少6周加4天,需要添加7类化学试剂和更换1次培养基。
这些缺点增加了利用程序性诱导分化技术诱导神经干细胞分化的材料成本、人工成本、操作复杂度和制备周期。
发明内容
本发明的目的是针对现有神经干细胞分化诱导技术中存在的种子细胞需要制备、分化成功验证较为复杂、分化周期长、操作步骤多等问题,选择适宜的种子细胞、降低神经干细胞分化诱导周期和成本,减少操作,给出简单易行的分化成功鉴定标准,提供一种调控神经干细胞分化的方法及其所用的生物制品。
本发明的上述目的可采用下列技术方案来实现:
本发明提供了一种调控神经干细胞分化的方法,所述调控方法是指通过激活信号通路或抑制分化抑制因子的方式,诱导神经干细胞的分化。
进一步的,上述的一种调控神经干细胞分化的方法,所述信号通路为核苷二磷酸激酶NDPK信号通路、Notch信号通路、Wnt/β-连锁蛋白信号通路或Pax-6信号通路。
进一步的,上述的一种调控神经干细胞分化的方法,所述分化抑制因子为Id1-Id4抑制因子中的一种或多种,优选地,所述分化抑制因子为ID3。
本发明的第二个目的是提供了一种用于调控神经干细胞分化的生物制品,所述生物制品是采用上述的调控方法诱导神经干细胞的分化。
进一步的,上述的生物制品,所述生物制品为甲基化转移酶、甲基化阅读蛋白和/或去甲基化酶。
进一步的,上述的生物制品,所述甲基化转移酶为能够催化mRNA上腺苷酸发生m6A修饰的m6A催化酶,优选地,m6A催化酶为METTL3/14、WTAP和/或KIAA1492。
进一步的,上述的生物制品,所述甲基化阅读蛋白为能够识别发生m6A修饰的碱基并能够激活下游调控通路的m6ARNA甲基化识别蛋白,优选地,m6ARNA甲基化识别蛋白为YTHDF1-3、YTHDC1-3、hnRNP和/或eIF3。
进一步的,上述的生物制品,所述去甲基化酶为能够对已发生m6A修饰的碱基进行去甲基化修饰的RNA去甲基化酶,优选地,RNA去甲基化酶为FTO和/或ALKBH5。
生物体内的许多蛋白质的含量都非常少,为了获得大量的目的蛋白,可通过基因工程技术将编码蛋白的核酸序列导入到某个宿主细胞,使其大量表达。这种将克隆化基因***合适载体后导入宿主细胞用于表达大量蛋白质的方法一般称为蛋白质表达技术。常用的表达***主要是大肠杆菌表达***。
表达操作的流程:原核表达载体的构建→转化到高效表达的宿主菌→目的蛋白的诱导表达→细菌的扩大培养→制备细菌蛋白粗提物→目的蛋白质的纯化。
本发明的第三个目的是提供了上述的调控神经干细胞分化的方法在促进神经干细胞分化中的应用,所述促进神经干细胞分化是指促进神经干细胞分化为神经元和星形胶质细胞。
本发明的第四个目的是提供了上述的用于调控神经干细胞分化的生物制品在促进神经干细胞分化中的应用,所述促进神经干细胞分化是指促进神经干细胞分化为神经元和星形胶质细胞。
C17.2神经干细胞是美国哈佛大学Snyder教授将V-myc基因通过MMLV逆转录病毒载体转入到小鼠小脑原代神经干细胞中,而构建的永生化细胞系。它具有神经干细胞的所有特征,是用来研究中枢神经***细胞再生的理想工具。研究报道,它可以经诱导分化成神经元、星形胶质细胞和少突胶质细胞。将C17.2神经干细胞体外扩增后,移植到Aβ1-40损伤大鼠海马,DAPI标记的NSCs在移植后6周,发生广泛迁移,大部分细胞分化为GFAP阳性的星形胶质细胞,少部分分化为NF200阳性神经元,突起长达胞体数倍,说明移植细胞可以选择性的分化为具有潜在功能的神经细胞。
神经干细胞增殖及分化的分子机制:神经干细胞的增殖分化是受基因调控的,外源性信号作用于细胞受体,影响信号转导和基因转录。内源性基因表达的方式受到调控,并且不同的基因作用于神经干细胞分化成熟的不同阶段,精准调控分化过程。神经元、星形胶质细胞和少突胶质细胞的产生受一组转录因子的调节,这些转录因子决定细胞的命运,并在神经***中指定亚型的身份。
分化抑制因子ID家族包括以下几种:
bHLH转录因子家族是一类重要的增强子和启动子DNA的结合蛋白,因其具有特征性的碱性螺旋-环-螺旋序列模式而得名,它以同源或异源二聚体形式与DNA上E-box序列-CANNTG结合而发挥作用。该家族成员参与多种细胞和组织分化发育的调控,在神经发育过程中起重要作用。调节神经干细胞分化的转录因子包括Mash1、NeuroD、Neurogenins(Ngnl,Ngn2)和Math家族等。它们参与神经干细胞分化的正调控,能够促进前体细胞的形成及增殖,促进神经元方向分化的比例,刺激神经细胞突触的形成。
ID是分化抑制因子,又称DNA结合抑制因子,属于螺旋-环-螺旋(HLH)转录因子家族成员之一,缺乏结合DNA的功能,通过与bHLH结合形成异二聚体而影响受bHLH调控的细胞特异性基因的表达,从而抑制DNA结合。ID蛋白通过与HES1蛋白直接相互作用,解除HES1的自反馈抑制,从而维持神经干细胞中HES1基因的表达。ID蛋白并不干扰HES1蛋白对其下游靶基因的抑制。ID1、ID2和ID3增加皮质神经干细胞的自我更新和增殖潜力,同时抑制神经元分化。ID蛋白干扰NeuroD/E47复合物与E-box序列的结合并抑制E-box介导的基因表达。NSCs中ID蛋白的过度表达增加了集落形成试验中神经球的数量和大小。ID3的表达在胚胎发育过程中呈动态调控状态,在细胞发育过程中,Id3基因大量表达于未分化的、增殖性细胞;随着细胞分化和成熟,Id3基因表达逐渐减少。大鼠胚胎的原位杂交显示,Id3基因在神经前体而非分化的神经细胞中高度表达。Id3消耗导致神经嵴前体的缺失和神经嵴衍生物的丢失,相反,Id3的过度表达会增加细胞增殖并导致神经嵴结构域的扩大。小鼠中对Id1和Id3的靶向破坏导致神经母细胞过早退出细胞周期并诱导神经特异性分化标记的表达。Id3参与调节神经分化相关BMP-SMAD信号通路。神经元细胞短暂接触BMP-2后便不能完成神经形成过程,同时上调Id1、Id3的表达。
其中,重点为m6A去甲基化修饰酶ALKBH5,AlkB同源蛋白5(AlkB homolog 5,ALKBH5)是Fe2+和α-酮戊二酸(α-ketoglutarate,a-KG)依赖的非血红素加氧酶,属于AlkB家族成员,定位在细胞核,是一种擦除m6A甲基化修饰的去甲基化酶。2013年,何川和杨运贵课题组首次证明了ALKBH5在体内和体外都具有去甲基化酶活力,他们利用RNA原位杂交技术鉴定发现,Alkbh5基因敲除能促进mRNA出核,证实m6A可能参与到mRNA的出核转运,是继FTO之后被发现的第二个mRNAm6A去甲基化酶,在mRNA的加工过程以及小鼠的***发育等生物过程中都起着重要的调控作用。
研究发现,在低压低氧环境下Alkbh5基因缺失会造成调控发育进程的基因m6A水平紊乱,加快RNA出核过程,成熟神经元数量减少,浦肯野细胞树突分支变短,星形胶质细胞放射状纤维也变短,从而导致小脑发育滞后,。此外还有研究显示ALKBH5/FTO共同调节Bcl2mRNA去甲基化,敲低Alkbh5基因会加剧神经元损伤,提示ALKBH5在脑缺血再灌注损伤中起着至关重要的作用。在星形胶质细胞中过表达circSTAG1可抑制ALKBH5向细胞核中转运,促使FAAH降解,显著减轻星形胶质细胞功能障碍和抑郁行为。
其它分化调节因子还包括:
a.BMP家族
TGF-β(transforming growth factorβ,TGF-β)超家族对神经***的发育、神经细胞的分化有着重要的作用。骨形态发生蛋白家族(bone morphogenic proteins family,BMP)是TGF-β超家族和生长因子家族中最大最重要的的成员之一,具有广泛的生物学活性,是一组具有类似结构的高度保守的功能蛋白,能够在体内诱导骨和腱样组织形成,在肢体生长、软骨内骨化、骨折早期及肌腱修复时表达,从而发挥其骨发生、骨诱导、骨修复及肌腱修复作用。BMP配体和受体亚单位在整个神经发育过程中都有表达,不同浓度的BMP和其他细胞因子共同促进在神经管时期不同类型神经细胞的分化。BMP2在脊髓损伤后表达上调,使神经干细胞向星形胶质细胞方向分化,而不向神经元方向分化。此外,骨形态发生蛋白对神经组织的形成以及在对神经元形成的类型和数量方面都起到一定的控制作用,并参与调节胚胎发育过程及造血祖细胞分化,某些成员还参与诱导细胞凋亡。Liu等运用实验胚胎学和遗传学方法最新证明了BMP在神经组织的形成以及在对神经元形成的类型和数量方面都起到一定的控制作用。
b.Notch信号***
Notch信号是一个在进化过程中高度保守的信号通路,广泛存在于无脊椎动物和脊椎动物的多个物种之中。它通过细胞间相互作用的方式精确地调节着细胞的分化、增殖和凋亡,在生物体发育过程中决定着细胞的不同命运。Notch信号通路在哺乳动物发育中参与神经发生过程,决定着中枢神经***祖细胞是选择继续增殖还是向神经元分化,因此它决定着包括神经细胞在内的许多细胞的命运。激活的Notch信号***是胚胎干细胞分化的抑制性通路,即当Notch被激活时,干细胞进行增殖;而Notch信号***活性被抑制时,干细胞进入分化程序,发育成为具有一定功能的细胞。Notch蛋白的作用为抑制干细胞向神经元方向分化,并促进向胶质细胞方向分化。Notch信号尤其在胚胎神经元发育和脑的发育过程中发挥重要作用,如果Notch信号紊乱将导致很多神经***疾病的发生,如常染色体显性遗传脑动脉病合并皮质下梗死和白质脑病(cerebral autosomal dominant arteriopathywith subcortical infarcts and leukoencephalopathy,CADASIL)、阿尔茨海默、皮层发育不良和脑肿瘤等。Notch信号能够通过放大并固化相邻细胞间的分子差异而影响细胞发育的命运,在不同的发育时间点上分别抑制了神经元和少突胶质细胞的分化,促进了神经干细胞向星形胶质细胞的分化。
c.Wnt家族
Wnt信号参与控制神经干细胞及神经嵴干细胞的增殖和决定其最终分化命运,对神经干细胞的增殖和分化均起关键调节作用。Wnt信号通路对神经干细胞的影响及其作用结果依赖于细胞的内在特性,从而决定神经于细胞如何对Wnt信号分子的反应。Wnt-1可通过缩短细胞***周期促进神经干细胞的增殖。同时,Wnt-1表达的严格时空调控对于中脑和后脑的正常发育至关重要。在体内和体外Wnt-3均通过激活Wnt/β—连锁蛋白信号通路促进海马神经前体细胞向神经元的分化,同时还与海马神经元的功能形成有密切的关系。
d.Pax家族
Pax基因家族是由一系列组织特异性的转录调控因子组成,这些转录因子都含有由6个α-螺旋构成的DNA结合区域,即所谓的配对域(paired domain),此外包括Pax-6基因在内的一些Pax基因还含有一个保守的同源框。Pax基因编码一组特异DNA序列结合转录因子,在胚胎发育,特别是神经***的发育中发挥着重要作用。研究表明Pax-6在室管膜前下区(anterior subventricular zone,SVZa)神经干细胞分化及迁移中发挥重要作用,敲除Pax-6基因后SVZa神经干细胞向嗅球迁移的数量减少,嗅脑的体积缩小,球周细胞层的TH阳性神经元的数目明显减少。
本发明的的特点及优点是:本发明技术方案过表达ALKBH5可促进神经干细胞分化为神经元和星形胶质细胞。ALKBH5可通过抑制分化抑制因子ID3的表达,促进神经干细胞分化为神经元和星形胶质细胞。选择C17.2细胞系作为神经干细胞分化的种子细胞,不需要制备,可以直接进行诱导分化,分化周期仅为4天,所需添加的试剂仅有2-3种,且为一次性加入,分化诱导期间不需要更换培养基,分化成功鉴定标志为NeuN(分化为神经元)和GFAP(分化为星形胶质细胞),鉴定过程简单明了,实施成本较低。本发明阐明了m6A去甲基化酶ALKBH5通过抑制分化抑制因子ID3的表达促进神经干细胞分化的具体机制,工作原理清晰,在后续神经干细胞分化工程任务中具有广泛的应用前景和实用价值。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为小鼠海马神经干细胞分化过程中ALKBH5表达变化,数据以平均值±标准差表示,n=3,*p<0.05。
图2为诱导C17.2细胞分化过程中ALKBH5表达变化,数据以平均值±标准差表示,n=3,*p<0.05。
图3为Si-ALKBH5对C17.2细胞分化的影响,数据以平均值±标准差表示,n=3。
图4为pcDNA-3.1-ALKBH5-myc质粒图。
图5为转染不同质量质粒对C17.2细胞NeuN、GFAP蛋白表达的影响;
A、转染Alkbh5过表达质粒对C17.2细胞ALKBH5蛋白表达的影响;B、转染Alkbh5过表达质粒对C17.2细胞GFAP、NeuN表达的影响,数据以平均值±标准差表示,n=3。
图6为C17.2细胞诱导分化以及抑制或增加ALKBH5合成后分化相关基因mRNA水平变化;
A、C17.2诱导分化后分化相关基因mRNA水平变化;B、转染Si-ALKBH548h后分化相关基因mRNA水平变化;C、转染过表达ALKBH548h后分化相关基因mRNA水平变化,数据以平均值±标准差表示,n=3,*p<0.05,**p<0.01,***p<0.001。
图7为C17.2诱导分化对Id3 mRNA稳定性的影响,数据以平均值±标准差表示,*p<0.05,**p<0.01。
图8为pGL3-promoter-Id3-3’UTR质粒模式图,
图9为Si-ALKBH5处理后Id3-3’UTR荧光素酶活性变化,数据以平均值±标准差表示,n=6,***p<0.001。
图10为MeRIP-qPCR检测m6A修饰的Id3 mRNA水平,数据以平均值±标准差表示,n=3,*p<0.05。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:
1.ALKBH5蛋白对神经干细胞分化的调节实验
1.1实验目的
以小鼠神经干细胞系C17.2为研究对象,通过增加或减少ALKBH5蛋白的合成研究ALKBH5对神经元标志蛋白——神经元特异性核蛋白(NeuN)和星形胶质细胞标志蛋白——胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)生成的影响。
1.2实验过程和结果
1.2.1小鼠海马神经干细胞分化过程中ALKBH5表达变化
a.实验方法
8周龄C57BL/6小鼠在动物房适应一周后,雄雌比例为1:3合笼,交配孕育生产后,分别对出生后1周、2周、3周、8周龄的小鼠取海马组织。
小鼠颈椎脱臼致死,剪开小鼠头皮和脑壳,暴露出脑组织;
海马组织被覆盖于大脑皮层之下,弯镊剥开脑皮层,暴露出海马组织。
轻轻剥离两侧海马,放入组织冻存管置于液氮罐中储存。
用RIPA提取组织蛋白,通过免疫印迹技术检测海马组织NeuN、GFAP、ALKBH5表达、内参蛋白GAPDH、α-tubulin表达。
b.实验结果
与1w小鼠海马组织相比,2w、3w、8w小鼠海马中NeuN、GFAP表达升高,ALKBH5表达同样增高。如图1所示。
c.实验小结
本实验初步证明,小鼠出生后,海马组织GFAP和NeuN表达上调,意味着神经干细胞发生了分化,期间伴随着ALKBH5表达上调。
1.2.2诱导C17.2细胞分化过程中ALKBH5表达变化
a.实验方法
C17.2细胞以DMEM/H+10%FBS+5%HS+1%PS培养体系培养,分别以DMEM/H+2.5%FBS+1.25%HS+1%PS和DMEM/H+2%FBS+1%HS+1%PS诱导分化4d,RIPA提取细胞蛋白,Western blotting检测NeuN、GFAP、ALKBH5蛋白和内参蛋白α-tubulin的表达。
b.实验结果
与正常培养组相比,2.5%FBS+1.25%HS和2%FBS+1%HS诱导分化4d后,NeuN、GFAP表达上调(p<0.05)。ALKBH5表达也上调。如图2所示。
c.实验小结
上述结果表明,C17.2神经干细胞诱导分化过程中,ALKBH5表达上调。
1.2.3抑制ALKBH5表达对神经干细胞分化的影响
a.实验方法
RNA干扰技术是减少蛋白合成的常用技术。通过细胞转染技术使RNA干扰剂siRNA与目标蛋白对应的信使RNA完全配对结合,抑制目标蛋白的转录和翻译,从而达到减少细胞内目标蛋白合成的目的。
NC和Si-ALKBH5粉末1OD以250μl DEPC水溶解至浓度为10μM的母液。
C17.2细胞以每孔2x105个细胞铺在六孔板,24h后进行细胞转染。
转染试剂RNAiMax按比例稀释到无血清培养基Opti-MEM,NC和SiRNA按比例稀释到无血清培养基Opti-MEM,两者等体积混匀,使得NC和Si-ALKBH5终浓度为100nM,静置5-10分钟,按照每孔250μl的混合液加入六孔板中。
转染8-10小时后换正常培养基培养4d。
RIPA提取细胞蛋白,Westernblotting检测NeuN、GFAP、ALKBH5蛋白和内参蛋白α-tubulin的表达。
其中,siRNA序列如下表2所示。
表2 alkbh5 SiRNA序列
b.实验结果
如图3所示。
c.实验小结
这些结果表明,抑制ALKBH5的合成,可导致神经干细胞NeuN、GFAP表达下降,从而抑制神经干细胞分化。
1.2.4增加ALKBH5合成对神经干细胞分化的影响
a.实验方法
基因过表达技术是增加细胞内蛋白合成的常用技术。通过细胞转染过表达质粒pcDNA-3.1-ALKBH5-myc,使其整合到细胞基因组上,可增强ALKBH5蛋白的转录和翻译,从而增加ALKBH5蛋白的合成。其中,myc是验证基因过表达成功与否的标志。Myc是一类小分子标签基因,可编码MYC蛋白,在哺乳动物体内没有此类蛋白。细胞转染过表达质粒pcDNA-3.1-ALKBH5-myc后,若检测到MYC蛋白,则表明基因过表达成功。
pcDNA-3.1和pcDNA-3.1-ALKBH5-myc菌液于添加15ul氨苄青霉素的15mL LB液体培养基中培养。摇菌12-16h后,提取质粒。
C17.2细胞以每孔2x105个细胞铺在六孔板,24h后进行细胞转染。
转染试剂Lipo3000按比例稀释到无血清培养基Opti-MEM,pcDNA-3.1、pcDNA-3.1-ALKBH5-myc质粒和p3000按比例稀释到无血清培养基Opti-MEM,两者等体积混匀,使得pcDNA-3.1终质量为5ug,pcDNA-3.1-ALKBH5-myc终质量每孔分别为1.25ug、2.5ug和5ug,静15分钟,按照每孔250每孔μl的混合液加入六孔板中。
转染48h后,利用RIPA提取细胞蛋白,Westernblotting检测NeuN、GFAP、ALKBH5蛋白和内参蛋白GAPDH的表达。
其中,pcDNA-3.1-ALKBH5-myc质粒如图4所示。
b.实验结果
与转染pcDNA3.1空载体组相比,转染pcDNA-3.1-ALKBH5-myc组均检测到ALKBH5表达上升,也检测到MYC蛋白的表达,ALKBH5质粒质量为2.5ug和5ug时,ALKBH5表达上升明显。
与转染pcDNA3.1空载体组相比,转染pcDNA-3.1-ALKBH5-myc组2.5ug和5ug组均检测到NeuN和GFAP表达上升。如图5所示。
c.实验小结
本实验证明,增加ALKBH5的合成可以上调C17.2细胞NeuN、GFAP蛋白的表达,从而促进神经干细胞的分化。
1.3研究小结
上述研究表明,神经干细胞分化过程中ALKBH5表达上调,ALKBH5可以促进神经干细胞分化。这提示,ALKBH5在调节神经干细胞分化过程中发挥了重要作用。
2.ALKBH5调节神经干细胞分化的机制研究
2.1研究目的
以小鼠神经干细胞C17.2为研究对象,抑制或增加ALKBH5蛋白表达,研究ALKBH5蛋白调节神经干细胞分化的分子机制。
2.2研究过程和结果
2.2.1 ALKBH5靶基因Id3的筛选及验证
a.实验方法
通过查阅文献筛选出与神经分化有关的并且可能受到ALKBH5调控的4个基因,即Tnik、Dab2、Sclt1和Id3。对C17.2细胞诱导分化后进行qRT-PCR验证,发现4个基因表达均有变化。通过抑制或增加ALKBH5合成,发现4个基因有不同的变化。
b.实验结果
qRT-PCR结果显示,C17.2诱导分化后,Tnik、Dab2的mRNA水平上调,Sclt1、Id3的mRNA水平下调。转染Si-ALKBH548h后,Tnik、Sclt1、Id3 mRNA水平上调,Dab2 mRNA无明显变化。转染过表达ALKBH548h后,Sclt1、Id3的mRNA水平下调,Tnik和Dab2的mRNA水平无明显变化。如图6所示。
c.实验小结
实验结果表明,C17.2诱导分化后Sclt1和Id3基因表达下调,敲低或过表达ALKBH5后,Id3 mRNA水平上调或下调。这提示,ALKBH5可以调节ID3表达水平。
2.2.2 C17.2诱导分化对Id3 mRNA稳定性的影响
a.实验方法
利用放线菌素D进行转录抑制实验。作用机制是放线菌素D嵌入DNA双链内与DNA上的鸟嘌呤基团结合,抑制RNA聚合酶活性,干扰转录过程。C17.2诱导分化后,加入5ug/mL的放线菌素D分别处理0,2,4,6h,qPCR检测Id3 mRNA的表达。
b.实验结果
与正常培养基培养组相比,诱导分化后,Id3 mRNA的半衰期由2.92h缩短到1.48h。如图7所示。
c.实验小结
神经干细胞诱导分化后Id3 mRNA的稳定性下降,半衰期缩短。
2.2.3 ALKBH5对Id3的m6A修饰的靶控关系
a.实验方法
以pGL3-promoter为载体构建含m6A位点的Id33’UTR区域的双荧光素酶报告基因质粒,NC或Si-Alkbh5与pGL3-promoter-Id3-3’UTR质粒共转染,每孔中均转染Renilla作为内参,进行双荧光素酶报告基因实验,化学发光仪检测荧光素酶活性。质粒模式图如图8所示。
m6A-IP-qPCR也叫MeRIP-qPCR,即利用m6A抗体富集甲基化修饰的RNA,然后通过qPCR技术对富集的RNA进行定量。
实验分为NC组和Si-Alkbh5组,细胞转染48h后,Trizol法提取RNA。
(1)将2x107个细胞及其对照组置于冰上,以细胞刮刀收集细胞,即刻使用RIP
lysis Buffer裂解细胞5分钟;
(2)将裂解细胞置于超声破碎仪中,隔冰超声破碎样本10秒钟;
(3)12000g,4℃离心10分钟,将样本上清液转移到新的1.5ml EP管中;
(4)使用RIPWash Buffer将磁珠清洗3次,与m6A抗体(SySy)置于旋转摇
床室温共孵育30分钟,使磁珠与抗体充分结合;
(5)孵育后的磁珠用RIPWash Buffer清洗3次,将未结合的抗体洗去,将磁珠
与裂解细胞置于旋转摇床4℃共同孵育过夜;
(6)将孵育了裂解细胞的磁珠用RIPWash Buffer温和清洗5次,将磁珠与蛋白
酶K在55℃孵育30分钟,使磁珠-蛋白-RNA复合物解体;
(7)将磁珠除去,取上清,使用酚:氯仿:异戊醇提取上清中的RNA,冻存在
-80℃或直接进行后续实验。
b.实验结果
与NC组相比,Si-ALKBH5处理后,pGL3-promoter-Id3-3’UTR荧光素酶活性显著上调。如图9所示。
与NC相比,Si-ALKBH5处理后,m6A修饰的Id3 mRNA水平显著上调。如图10所示。
c.实验小结
ALKBH5可以通过调节m6A去甲基化负向调节影响Id3基因表达。
2.3研究小结
神经干细胞分化期间伴随着分化抑制基因Id3 mRNA稳定性下降所致的表达下调,ALKBH5可以通过调节m6A去甲基化抑制Id3基因表达。
ALKBH5可以促进神经干细胞分化为神经元和星形胶质细胞。其机制是通过促进分化抑制蛋白ID3的m6A发生去甲基化,导致ID3水平下降,解除对神经干细胞分化的抑制,从而发挥其调节神经干细胞分化的作用。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.一种调控神经干细胞分化的方法,其特征在于,所述调控方法是指通过激活信号通路或抑制分化抑制因子的方式,诱导神经干细胞的分化。
2.根据权利要求1所述的一种调控神经干细胞分化的方法,其特征在于,所述信号通路为核苷二磷酸激酶NDPK信号通路、Notch信号通路、Wnt/β-连锁蛋白信号通路或Pax-6信号通路。
3.根据权利要求1所述的一种调控神经干细胞分化的方法,其特征在于,所述分化抑制因子为Id1-Id4抑制因子中的一种或多种,优选地,所述分化抑制因子为Id3。
4.一种用于调控神经干细胞分化的生物制品,其特征在于,所述生物制品是采用权利要求1-3任意一项所述的调控方法诱导神经干细胞的分化。
5.根据权利要求4所述的生物制品,其特征在于,所述生物制品为甲基化转移酶、甲基化阅读蛋白和/或去甲基化酶。
6.根据权利要求5所述的生物制品,其特征在于,所述甲基化转移酶为能够催化mRNA上腺苷酸发生m6A修饰的m6A催化酶,优选地,m6A催化酶为METTL3/14、WTAP和/或KIAA1492。
7.根据权利要求5所述的生物制品,其特征在于,所述甲基化阅读蛋白为能够识别发生m6A修饰的碱基并能够激活下游调控通路的m6ARNA甲基化识别蛋白,优选地,m6ARNA甲基化识别蛋白为YTHDF1-3、YTHDC1-3、hnRNP和/或eIF3。
8.根据权利要求5所述的生物制品,其特征在于,所述去甲基化酶为能够对已发生m6A修饰的碱基进行去甲基化修饰的RNA去甲基化酶,优选地,RNA去甲基化酶为FTO和/或ALKBH5。
9.权利要求1-3任一所述的调控神经干细胞分化的方法在促进神经干细胞分化中的应用,其特征在于,所述促进神经干细胞分化是指促进神经干细胞分化为神经元和星形胶质细胞。
10.权利要求4-8任一所述的用于调控神经干细胞分化的生物制品在促进神经干细胞分化中的应用,其特征在于,所述促进神经干细胞分化是指促进神经干细胞分化为神经元和星形胶质细胞。
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