CN117025683A - 一种利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法 - Google Patents
一种利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法 Download PDFInfo
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Abstract
本发明公开了一种利用Snurf‑Snrpn来促进诱导多能干细胞定向神经分化的方法,其方法为:第一步、利用mRNA Snurf‑Snrpn提高小鼠体细胞重编程效率,获得Snurf‑Snrpn‑iPSC细胞系;第二步、诱导Snurf‑Snrpn‑iPSC细胞系定向神经元分化;有益效果:提高了体细胞重编程的效率;提高了体外定向诱导iPSC向神经元细胞分化的效率,能够提高32%左右。为优化或建立iPSC向神经元细胞高效、定向分化方案奠定理论基础,推动诱导干细胞产品治疗神经退行性疾病的临床转化。
Description
技术领域
本发明涉及一种定向神经分化的方法,特别涉及一种利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法。
背景技术
目前,神经退行性疾病是一类可导致感觉丧失、运动功能丧失和记忆衰竭等症状的难治性疾病,常见的代表性疾病有创伤性脑损伤(TBI)、脊髓损伤(SCI)、中风(Stroke)、阿尔茨海默病AD)及帕金森综合征(PD)等。内源性神经细胞的可修复性不足是治疗该类疾病的重要障碍。传统治疗方法虽能延缓疾病进展,但局限性明显。而神经干细胞移植作为一种潜在的新型治疗方式能够有效促进神经细胞的功能恢复及组织再生,在神经退行性疾病的治疗应用方面前景广阔。
由于高龄、失能的人群逐渐增多,给社会发展带来了沉重的负担,因此对于这部分人群的治疗,改善其生活质量已成为迫在眉睫的事情。iPSC因可由体细胞重编程获得,成为能无限提供分泌神经干细胞来源的种子细胞,备受瞩目。但尽管已经有不少研究可以在体外诱导干细胞分化获得前脑神经元,但是通常存在耗时长、神经元纯度不高、以及成熟度不够等问题。因此,在体外快速、高效地获得成熟前脑神经元是近几年国内外的研究重点及趋势。这不论对于神经系统疾病新型药物筛选,或是推进细胞替代治疗的临床转化都意义重大。
发明内容
本发明的目的是为了解决体外诱导干细胞分化获得前脑神经元所存在的耗时长、神经元纯度不高以及成熟度不够等诸多问题,而提供的一种利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法。
本发明提供的利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法,其方法包括的步骤如下:
第一步、利用mRNA Snurf-Snrpn提高小鼠体细胞重编程效率,获得Snurf-Snrpn-iPSC细胞系,具体步骤如下:
步骤一、构建Snurf-Snrpn过表达质粒;
步骤二、慢病毒包装、感染小鼠MEF原代细胞,已事先转入OSKM因子;同时设VectorCT质粒对照组,及空白对照组;
步骤三、在细胞培养液中加入DOX诱导OSKM因子表达,重编程启动;
步骤四、确定形成iPSCs克隆的多能性,并计数比较三组之间NANOG阳性克隆数;
步骤五、取重编程成功的iPSCs克隆,在已经铺好的滋养层细胞上进行培养,获得Snurf-Snrpn-iPSC细胞系,细胞状态较好时进行冻存;
第二步、诱导Snurf-Snrpn-iPSC细胞系定向神经元分化,具体步骤如下:
步骤一、Snurf-Snrpn-iPSC细胞的培养;
步骤二、用悬滴法诱导拟胚体EBs形成;
步骤三、将拟胚体放到用0.1%的明胶包被的12孔细胞培养板中继续培养;
步骤四、收集各个时间点的细胞,检测多能性指标,三胚层分化指标的变化;
步骤五、用免疫荧光染色检测EB分化为神经元细胞的情况。
本发明的有益效果:
本发明提供的利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法采用过表达Snurf-Snrpn的方式,提高了体细胞重编程的效率;提高了体外定向诱导iPSC向神经元细胞分化的效率,能够提高32%左右。为优化或建立iPSC向神经元细胞高效、定向分化方案奠定理论基础,推动诱导干细胞产品治疗神经退行性疾病的临床转化。
附图说明
图1为本发明所述的Snurf-Snrpn结构示意图。
其中Snurf-Snrpn基因位于小鼠7号染色体C区域,是一种双顺反子基因,编码两种不同的蛋白质;由外显子1-3编码SNURF,一种功能未知的多肽;下游外显子4-10编码SNRPN,一种参与mRNA剪接的剪接体蛋白;E1-E10;外显子1-10。
图2为本发明所述的Snurf-Snrpn过表达效果示意图。
其中WT:野生型细胞,即小鼠MEF原代细胞;Vector-CT:转入Vector对照质粒的小鼠MEF原代细胞;Snurf-Snrpn:转入Snurf-Snrpn过表达质粒的小鼠MEF原代细胞。
图3为本发明所述的重编程实验结果示意图。
其中可见过表达Snurf-Snrpn组的细胞形成克隆数量明显高于两个对照组。WT:野生型细胞,即小鼠MEF原代细胞;Vector-CT:转入Vector对照质粒的小鼠MEF原代细胞;Snurf-Snrpn:转入Snurf-Snrpn过表达质粒的小鼠MEF原代细胞。
图4为本发明所述的诱导Snurf-Snrpn-iPSC细胞系定向神经元分化流程图。
其中第一阶段为EB形成期,第二阶段为诱导EB向神经元细胞分化期。
图5为Snurf-Snrpn-iPSC细胞系定向神经元分化结果示意图。
其中相对于对照组,Snurf-Snrpn-iPSC细胞系向外胚层分化比例明显增加,神经元细胞染色提示Snurf-Snrpn-iPSC组神经元阳性细胞形成较多。WT:野生型细胞,即小鼠MEF原代细胞;Vector-CT:转入Vector对照质粒的小鼠MEF原代细胞;Snurf-Snrpn:转入Snurf-Snrpn过表达质粒的小鼠MEF原代细胞。
具体实施方式
请参阅图1至图5所示:
实施例1、构建Snurf-Snrpn过表达质粒并转染目的细胞
1、通过PCR体外扩增Snurf-Snrpn
设计引物
JH5594 5’-TCGGGCGCCAGATATCggcaaaaatgtgcgcatgtgcagccat-3’
JH5595 5’-agAatcgaaGTCGACtcttcttcaggaaaatcattttataa-3’
克隆Snurf-Snrpn所需PCR体系(50μL)
PCR条件
将PCR产物跑核酸胶,验证大小,大小正确则回收进行下一步实验。
2、酶切:将Snurf-Snrpn目的片段及lenti-RsRED目的载体用同样的快速限制性内切酶SalI和EcoRV分别切开(酶切体系如下),并分别跑胶回收。
载体酶切体系(20μL)
3、连接:
将上一步回收的Snurf-Snrpn全长片段与载体用T4连接酶22℃连接0.5h。
连接体系如下:
4、转化、扩增培养、鉴定:
将合成的质粒转入到大肠杆菌感受态细胞中,进行扩增培养后涂布于LB固体培养基上,待长出菌落后进行挑菌PCR鉴定,最后将鉴定阳性的菌落送测序确定Snurf-Snrpn序列全部正确,说明Snurf-Snrpn过表达质粒合成成功。
5、慢病毒包装、感染目的细胞及筛选:
利用慢病毒包装技术在细胞中进行病毒的包装,包装好的病毒颗粒分泌到细胞外的培养基中,过滤后可以直接用于宿主细胞的感染,目的基因进入宿主细胞之后,经过反转录,整合到基因组,从而高水平的表达目的基因。
(1)、慢病毒包装:
①我们采用293T细胞进行病毒包装,6孔板培养293T细胞,当细胞长至70%左右时进行慢病毒包装,将目的质粒(过表达及对照质粒)、慢病毒包装质粒psPAX2及pMD2G按恰当比例混合好加入100μL Opti-MEM中混合均匀,同时将1μL浓度为5μg/μL的PEI加入100μLOpti-MEM中混合均匀,两种混合液同时于室温静置5分钟后将两种液体混合均匀,于室温静置20min,接下来进行病毒包装,将以上混合液均匀点滴至293T细胞的培养液中后将细胞放回培养箱中继续培养。
②6-8h后更换培养液,让细胞在正常DMEM培养液中继续培养。从更换培养液之后开始计时,分别收集24h、48h、72h的病毒上清并用0.45μM滤膜过滤,过滤后直接感染目的细胞,剩余的病毒上清分装存于-80℃冰箱备用。
(2)、感染目的细胞及筛选:
①将待转染的MEF接种于6孔板培养板,待细胞生长至50%-60%时可用于感染病毒。因MEF转染效率较低,为确保转染效率,连续进行转染3次,利用转染后是否有过表达质粒所特有的红色荧光初步判断转染效率,取部分细胞进一步应用RT-qPCR验证转染效率。
②将新鲜制备好的病毒液按一定比例均匀点滴至目的细胞的培养液中,可同时加入Polybrene作为助转剂(使得Polybrene在整个细胞配液也中终浓度为8ng/μL)。
③待病毒作用24h后,弃去含有病毒的培养液,更换为正常的培养液,将原液浓度10mg/mL的嘌呤霉素(Puromycin)稀释100倍配制成0.1μg/μL的工作液。取10μL嘌呤霉素工作液加入培养液中,使嘌呤霉素从浓度为0.5μg/mL开始进行筛选。
④观察细胞死亡情况,增加嘌呤霉素浓度至2μg/mL,细胞仍不能被嘌呤霉素杀死时,耐药克隆确认。
实施例2、诱导重编程,获得Snurf-Snrpn-iPSC细胞系:
重编程实验所用细胞为已事先转入OSKM因子的小鼠MEFs原代细胞,这种细胞以DOX为开关,在没有DOX的培养液中,OSKM不表达,不能进行重编程,当加入DOX后,OSKM表达,启动重编程进程。
(1)重编程前准备:分别取转染了Snurf-Snrpn过表达质粒,转染了VectorCT质粒及空白MEF细胞,按密度为每孔2×104分盘至12孔细胞培养板中用于重编程实验。
(2)重编程实验:在细胞培养液中加入DOX诱导OSKM因子表达,重编程启动,每天观察MEFs形态变化,进行拍照记录,大约在诱导开始的第12天,观察到首个iPSCs克隆出现,在诱导的第14天左右,大部分克隆皆已形成,且有部分克隆已开始出现死亡细胞,停止重编程实验,对细胞进行NANOG免疫荧光染色,并取部分细胞提取RNA进行性逆转录及PCR鉴定,确定形成iPSCs克隆的多能性,并计数比较三组之间NANOG阳性克隆数。
(3)iPSCs培养:取重编程成功的iPSCs克隆,在已经铺好的滋养层细胞上进行培养,细胞状态较好时进行冻存,获得Snurf-Snrpn-iPSC细胞系,并取部分细胞提取RNA,逆转录为cDNA进行PCR验证几个比较重要的多能因子Oct4,Sox2,Nanog来明确iPSCs的多能性,并比较三个实验组之间多能性基因的表达差异。
实施例3、诱导Snurf-Snrpn-iPSC细胞系定向神经元分化:
在DMSO中准备10mm的维A酸原液,并分装到1.5mL光保护的微生物管中。该溶液在-80℃下稳定长达两周。保护它不受光线照射。
(1)将生长至60-70%的胚胎干细胞消化后用不含白血病抑制因子(leukemiainhibitory factor,LIF)的含0.5μM维A酸的knockout培养液重悬,计数后将细胞稀释至密度为3×104mL。
(2)准备足够的10cm培养皿,加入10mL新鲜配置的PBS,将培养皿的盖子倒置,分别点滴20-30μL上述稀释好的胚胎干细胞,每个盖子可点滴20滴左右。轻轻将盖子扣在皿底,放到37℃,5%CO2孵箱培养4天左右。期间收集D0、D2及D4细胞提取RNA。
(3)用1ml 0.1%的明胶将12孔细胞培养板进行包被,在37℃,5%CO2孵箱中放置2h以上。
(4)用200μL吸管头,一个接一个地收获3-4天生长的挂滴EBs,播种在明胶包被的12孔细胞培养板中,每孔20EBs。继续用不含LIF的含0.5μM维A酸的knockout培养液培养,每2-3天更换培养液。
(5)用PBS洗涤细胞后,在PBS中加入4%多聚甲醛固定10分钟。用PBS洗涤3次,去除漂浮的细胞碎片。
(6)冰上,0.5%Triton X-100透化细胞10min。
(7)PBS洗3遍,加入1%BSA,室温封闭1h。
(8)一抗(1:500),4℃封闭过夜。
(9)PBS洗3遍,二抗(1:1000)室温孵育1h。
(10)PBS洗3遍,DAPI37℃孵育5-7min。
表1.PCR引物
Claims (1)
1.一种利用Snurf-Snrpn来促进诱导多能干细胞定向神经分化的方法,其特征在于:其方法包括的步骤如下:
第一步、利用mRNA Snurf-Snrpn提高小鼠体细胞重编程效率,获得Snurf-Snrpn-iPSC细胞系,具体步骤如下:
步骤一、构建Snurf-Snrpn过表达质粒;
步骤二、慢病毒包装、感染小鼠MEF原代细胞,已事先转入OSKM因子;同时设VectorCT质粒对照组,及空白对照组;
步骤三、在细胞培养液中加入DOX诱导OSKM因子表达,重编程启动;
步骤四、确定形成iPSCs克隆的多能性,并计数比较三组之间NANOG阳性克隆数;
步骤五、取重编程成功的iPSCs克隆,在已经铺好的滋养层细胞上进行培养,获得Snurf-Snrpn-iPSC细胞系,细胞状态较好时进行冻存;
第二步、诱导Snurf-Snrpn-iPSC细胞系定向神经元分化,具体步骤如下:
步骤一、Snurf-Snrpn-iPSC细胞的培养;
步骤二、用悬滴法诱导拟胚体EBs形成;
步骤三、将拟胚体放到用0.1%的明胶包被的12孔细胞培养板中继续培养;
步骤四、收集各个时间点的细胞,检测多能性指标,三胚层分化指标的变化;
步骤五、用免疫荧光染色检测EB分化为神经元细胞的情况。
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