CN107828826A - 一种体外高效获得神经干细胞的方法 - Google Patents
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Abstract
本发明涉及细胞生物学技术领域，具体来说是一种体外高效获得神经干细胞的方法，A、通过CRISPR/Cas9系统构建拥有Pax6‑GFP报告系统的胚胎干细胞细胞系；B、Pax6‑GFP ES报告系统在体外和体内指示神经分化；C、Pax6‑GFP报告系统富集纯化神经干细胞；本发明使用CRISPR/Cas9基因编辑工具构建了具有Pax6‑GFP荧光报告系统的细胞系，并通过这个细胞系实现了体外神经干细胞的快速、高效获得。
Description
技术领域
本发明涉及细胞生物学技术领域，具体来说是一种体外高效获得神经干细胞的方法。
背景技术
由于一些神经退行性疾病的病因是某些神经元的缺失，因此干细胞疗法对于治疗此类疾病有很大的潜在价值，神经干细胞就是其中不可或缺的一环。如何在体外快速获得神经干细胞是科学家们一直探讨的问题。现有体外分化神经干细胞的方法主要有三种：EB悬浮培养不定向分化到神经干细胞；胚胎干细胞与特定基质细胞共培养；在特定生长因子条件下单层贴壁培养。这三种方法有一个共同的缺点，即需要实验者的经验丰富，操作的主观性太强。
发明内容
本发明的目的是提供一种体外高效获得神经干细胞的方法。
为实现上述目的，本发明采用的技术方案是：一种体外高效获得神经干细胞的方法，包括如下步骤：
A、通过CRISPR/Cas9系统构建拥有Pax6-GFP报告系统的胚胎干细胞细胞系；
B、Pax6-GFP ES报告系统在体外和体内指示神经分化；
C、Pax6-GFP报告系统富集纯化神经干细胞。
进一步的，所述步骤A具体包括：
A1、设计供体质粒，包含左同源臂、增强型绿色荧光蛋白、由SV40启动子启动的新霉素抗性基因和右同源臂，并在增强型绿色荧光蛋白之前插入自剪接多肽，将整个元件插入到Pax6基因17号外显子后面，替换掉原有的终止密码子，使Pax6和增强型绿色荧光蛋白共表达(图1所示)；
A2、在pax6基因附近设计了两种sgRNA，以引起目标基因组的双链断裂，造成基因组上的缺口，便于供体质粒的整合；
A3、在用Cas9-sgRNA质粒和供体质粒转染两天后，用流式细胞仪进行分选Cas9-GFP阳性细胞；
A4、将分选得到的细胞进一步在含有G418的ESCs培养基中培养以选择抗性克隆，用3对引物通过PCR鉴定随机挑选的亚克隆(图2所示)，挑选纯合子作为拥有Pax6-GFP报告系统的纯合的ES细胞系，即6#细胞，命名Pax6GFP/GFP。
进一步的，步骤A1中，两种所述sgRNA为sgRNA1：CCCGGTGTGGATGCGGATAT；sgRNA2：AGGCCTCTTTTGGTATTCCA。
进一步的，步骤A4中，引物序列为：
P1 forward：TTCCACCCAGCCCCATTTTG；
P1 reverse:AGTGTGTGTTGTCCCAGGTTC；
P2 forward:ACTGTTCCCAAGGTCCCTACAT；
P2 reverse:CTTGTGGCCGTTTACGTCGC；
P3 forward:TCGCCTTCTATCGCCTTCTTGA；
P3 reverse:TGTGTCTGTGCTCATTATTCCTTG。
进一步的，所述步骤B具体包括：
B1、选择6#细胞系通过拟胚体的方法进行神经分化；
B2、在培养过程中，利用流式细胞仪进行分析从6天分化到12天的GFP动态变化，选择分化第7天的细胞混合物分选神经干细胞并收集，收细胞后进一步在NSC培养基中培养，其具有神经干细胞。
进一步的，所述步骤C具体包括：
C1、将携带Pax6-GFP的小鼠胚胎干细胞悬浮培养3天，然后在神经培养基粘附培养4天；
C2、在分化7天后，将GFP阳性细胞通过流式细胞仪分选回来并铺在具有NSC培养基的纤维连接蛋白预铺的培养皿中；
C3、进行免疫荧光染色，确定分选的GFP阳性细胞是否是神经干细胞，确认分选都细胞表达Pax6、Nestin和Sox1；在未处理的悬浮培养皿中，它们自发聚集形成紧密的神经球，其也表达神经干细胞的特异性标记基因。
本发明的有益技术效果是：本发明使用CRISPR/Cas9基因编辑工具构建了具有Pax6-GFP荧光报告系统的细胞系，并通过这个细胞系实现了体外神经干细胞的快速、高效获得。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术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性的前提下，还可以根据这些附图获得其他的附图。
图1是本发明供体质粒结构示意图；
图2是本发明的基因型检测示意图。
具体实施方式
下面将结合本发明实施例，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作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。
1、质粒构建
使用CRISPR设计网站(www.crispr.mit.edu)设计sgRNA1和sgRNA2(sgRNA1：CCCGGTGTGGATGCGGATAT；sgRNA2:AGGCCTCTTTTGGTATTCCA)，并克隆至PX461载体(Addgene)中。具体方法为，对PX461载体用BbsI(Thermo)进行酶切，然后对酶切产物进行纯化回收。对寡聚核苷酸sgRNA1和sgRNA2磷酸化退火。将退火的寡核苷酸连接到上述酶切回收完的载体中。Pax6-T2A-eGFP-neoR质粒含有5'臂，T2A-GFP，neoR和3'臂(图1)。5'臂长度为1245bp，3'臂长度为1436bp，3'和5'从基因组DNA PCR扩增而来。在供体质粒中，将T2A-eGFP序列与Pax6的最后一个密码子融合。
2、细胞培养和转染
将遗传背景为129Sv/Jae的mESC在R1+2i培养基中培养(R1+2i培养基配方为添加了15％胎牛血清(FBS)，1％非必需氨基酸，0.1mMβ-巯基乙醇，100mg/mL青霉素-链霉素的DMEM/F12，再加1mM丙酮酸钠，1单位/mL白血病抑制因子(LIF)，40mM PD0325901和120mMCHIR99021)。对小鼠胚胎成纤维细胞(MEF)用灭活的丝裂霉素-C处理成为饲养层细胞(feeder)。每2天用0.25％胰蛋白酶/EDTA对细胞进行传代。进行分化时，将细胞在EB培养基和N2B27培养基中培养，EB培养基为不含LIF的R1培养基。NSC在具有10ng/Ml EGF和bFGF的N2B27培养基中培养。
为了得到Pax6-GFP报告系统的mESC细胞系，我们使用Lipofectamine LTX试剂盒(Invitrogen)进行脂质体转染。具体方法如下：用2μg sgRNA1-Cas9n，2μg sgRNA2-Cas9n和4μg Pax6-GFP供体质粒转染约1×10 6个ESC。GFP转染后36h后通过流式细胞仪分选GFP阳性细胞。用250mg/mL G418筛选阳性细胞，时间为7天。然后挑选存活的亚克隆用于基因型鉴定。
3、神经分化
将小鼠胚胎干细胞克隆用0.25％胰蛋白酶/EDTA消化成单细胞，用于NPC分化。将这些细胞种在含有ES培养基的0.2％明胶预铺的培养皿中30分钟以除去饲养层细胞。然后，将mESC悬浮为密度为5×10 5个细胞/mL，种在含有EB培养基的未铺底的培养皿中。在第3天，将长成的拟胚体(EB)接种在预先用纤维连接蛋白铺底的皿中，并在N2B27培养基中培养。在分化7天后，用BD FACS Aria III分选GFP阳性细胞。数据采用FlowJo V10软件分析。
4、神经元分化
为了验证我们由报告系统得到的神经干细胞具有分化潜能，将NPC以1×104个细胞/孔的密度接种到PDL/层粘连蛋白铺底的24孔板中，培养基为添加了10ng/mL脑源性神经营养因子(BDNF)和10ng/mL神经降压素(NT-3)的N2B27培养基。两周后，分化的细胞具有少突神经胶质标记O4和神经元标记NeuN和TH的免疫活性。为了获得星形胶质细胞，我们将NSC在含有1％FBS和10ng/mL骨成型蛋白质4(BMP4)的N2B27培养基中培养5-7天，然后进行GFAP和Map2染色。
5、免疫荧光染色
对于免疫荧光，用4％多聚甲醛(PFA)将细胞固定10分钟，在室温下用磷酸盐缓冲盐水(PBS)洗涤一次。在用PBS洗涤后，在室温下用0.3％Triton X-100(Sigma)封闭一小时。然后在用PBS稀释的3％牛血清白蛋白(BSA)中将细胞封闭1小时。神经球的固定：在室温下用4％PFA固定神经球20min，用PBS洗涤，放置3min，反复洗涤3次。用0.3％Triton X-100将固定的神经球在室温下温育5min，洗涤3次，然后在含有3％BSA的PBS中封闭30min，用PBS洗涤并放置3min。将该洗涤程序重复三次。
一抗和二抗用含有1％BSA和0.3％Triton X-100的PBS稀释。样品与一抗在4℃孵育过夜，然后用PBS洗涤3次，并在室温下与二抗孵育1h。细胞核在室温下用DAPI染色5-10min。使用Leica TCS SP8显微镜收集图像。
6、定量PCR(qPCR)
使用Trizol溶液(Invitrogen)按照从ESC或NPC中提取总RNA，并使用PrimeScriptTM RT试剂盒(Takara)合成cDNA。实时RT-PCR使用SYBR Premix EX Taq kit(Takara)在ABIQuantStudioTM 6Flex机上进行。样品以GAPDH为参照扩增。
7、嵌合动物的获得
从小鼠交配2天后的雌性CD1小鼠收集4细胞期的胚胎，并使用10至12个Pax6-GFP的胚胎进行显微注射。将重建的胚胎在KSOM/AA中于37℃在5％CO2中孵育30min，并转移到假孕的CD1雌性小鼠的子宫中。E10.5和E13.5时收集怀孕小鼠嵌合胚胎，并通过荧光显微镜(尼康)鉴定绿色荧光蛋白的表达情况。
8、电生理分析
将NSC接种在铺有PDL/层粘连蛋白的35mm培养皿上。14-21天后这些细胞分化为运动神经元。将该皿放置在40×物镜的显微镜上。培养基改为细胞外溶液，成分为119mMNaCl，26.2mM NaHCO3，11mM葡萄糖，2.5mM KCl，2.5mM CaCl2，1.3mM MgCl2和1mM K2HPO4。用于记录全细胞动作电位的电极液成分为130mM KCl，10mM NaCl，2mM MgCl2，10mM Hepes，0.5mM EGTA，0.16mM CaCl2，用1M KOH调节pH至7.2。在电压钳模式下记录细胞电流，基础保持电位为-70mV；在-90至+30mV的电压范围内，以10mV增量递送，TTX的浓度为100nM，使用PClampfit 10.2收集和分析数据。
最后所应说明的是：以上实施例仅用以说明而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应该理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.一种体外高效获得神经干细胞的方法，其特征在于，包括如下步骤：
A、通过CRISPR/Cas9系统构建拥有Pax6-GFP报告系统的胚胎干细胞细胞系；
B、Pax6-GFP ES报告系统在体外和体内指示神经分化；
C、Pax6-GFP报告系统富集纯化神经干细胞。
2.根据权利要求1所述的方法，其特征在于，所述步骤A具体包括：
A1、设计供体质粒，包含左同源臂、增强型绿色荧光蛋白、由SV40启动子启动的新霉素抗性基因和右同源臂，并在增强型绿色荧光蛋白之前插入自剪接多肽，将整个元件插入到Pax6基因17号外显子后面，替换掉原有的终止密码子，使Pax6和增强型绿色荧光蛋白共表达；
A2、在pax6基因附近设计了两种sgRNA，以引起目标基因组的双链断裂，造成基因组上的缺口，便于供体质粒的整合；
A3、在用Cas9-sgRNA质粒和供体质粒转染两天后，用流式细胞仪进行分选Cas9-GFP阳性细胞；
A4、将分选得到的细胞进一步在含有G418的ESCs培养基中培养以选择抗性克隆，用3对引物通过PCR鉴定随机挑选的亚克隆，挑选纯合子作为拥有Pax6-GFP报告系统的纯合的ES细胞系，即6#细胞，命名Pax6GFP/GFP。
3.根据权利要求2所述的方法，其特征在于，步骤A1中，两种所述sgRNA为sgRNA1：CCCGGTGTGGATGCGGATAT；sgRNA2：AGGCCTCTTTTGGTATTCCA。
4.根据权利要求2所述的方法，其特征在于，步骤A4中，引物序列为：
P1forward：TTCCACCCAGCCCCATTTTG；
P1reverse:AGTGTGTGTTGTCCCAGGTTC；
P2forward:ACTGTTCCCAAGGTCCCTACAT；
P2reverse:CTTGTGGCCGTTTACGTCGC；
P3forward:TCGCCTTCTATCGCCTTCTTGA；
P3reverse:TGTGTCTGTGCTCATTATTCCTTG。
5.根据权利要求1所述的方法，其特征在于，所述步骤B具体包括：
B1、选择6#细胞系通过拟胚体的方法进行神经分化；
B2、在培养过程中，利用流式细胞仪进行分析从6天分化到12天的GFP动态变化，选择分化第7天的细胞混合物分选神经干细胞并收集，收细胞后进一步在NSC培养基中培养，其具有神经干细胞。
6.根据权利要求1所述的方法，其特征在于，所述步骤C具体包括：
C1、将携带Pax6-GFP的小鼠胚胎干细胞悬浮培养3天，然后在神经培养基粘附培养4天；
C2、在分化7天后，将GFP阳性细胞通过流式细胞仪分选回来并铺在具有NSC培养基的纤维连接蛋白预铺的培养皿中；
C3、进行免疫荧光染色，确定分选的GFP阳性细胞是否是神经干细胞，确认分选的细胞表达Pax6、Nestin和Sox1；在未处理的悬浮培养皿中，它们自发聚集形成紧密的神经球，其也表达神经干细胞的特异性标记基因。
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