CN117143880B - 一种etv2的orf序列以及一种内皮细胞的制备方法 - Google Patents
一种etv2的orf序列以及一种内皮细胞的制备方法 Download PDFInfo
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Abstract
本发明提供了一种ETV2的ORF序列以及一种内皮细胞的制备方法,属于内皮细胞开发技术领域。本发明中ETV2的ORF序列如SEQ ID NO.1所示,能够高效表达ETV2蛋白,用于翻译能够得到更多的ETV2蛋白,表达效率更高,用于将干细胞分化为内皮细胞能够显著提升转化效率。本发明提供的内皮细胞的制备方法,制备效率高、且临床应用风险较低。因此解决了现有技术中无法安全高效地将干细胞分化为内皮细胞的问题。本发明能够高效快速地实现内皮细胞的大量制备,满足细胞命运重编程和内皮分化的需求。本发明提供的制备方法为内皮细胞直接注射到缺血部位、促进血管新生、改善组织灌注情况提供了稳定的细胞来源。
Description
技术领域
本发明涉及内皮细胞开发技术领域,尤其涉及一种ETV2的ORF序列以及一种内皮细胞的制备方法。
背景技术
内皮细胞(Endothelial Cells,ECs)是构成血管和淋巴管的重要组成部分。ECs参与并调节多个生物学过程,如器官和组织间的互作,血管对外周血中物质的通透性,组织的血液灌注情况,炎症的发生发展以及血栓的形成。ECs的功能紊乱会导致凝血-纤溶平衡的失调,进而使血管内形成血栓,阻碍了血液流动,最终导致组织的缺血坏死,严重影响患者的生活质量。若梗阻部位位于脑血管或冠状动脉,则患者有梗死风险。因此,如何促使血管再通,恢复组织灌注是解决缺血性疾病的关键。
目前临床常用的治疗缺血性疾病的方法为溶栓或支架治疗,但由于ECs自身的功能紊乱未得到改善,部分患者治疗后出现再次梗阻,疗效不佳。干细胞技术近年来的发展为缺血性疾病的治疗提供了新的方案。通过将干细胞来源的内皮细胞(Stem-Cell-DerivedEndothelial Cells,SC-ECs)直接注射到缺血部位,能够有效促进血管新生,改善组织灌注情况。但如何在体外细胞培养中安全高效地将干细胞分化为内皮细胞仍是领域内亟待解决的问题。
现有技术公开了转录因子E26转化-特异性变体2(E26 Transformation-SpecificVariant 2,ETV2)在内皮谱系分化中发挥着至关重要的作用,其作为先驱转录因子,具有将体细胞及多能干细胞重编程为内皮细胞的能力。研究表明,在小鼠胚胎成纤维细胞和人多能干细胞中过表达ETV2能成功分化出ECs,持续表达7天后分化效率近100%。显然,ETV2过表达分化内皮细胞的策略在分化效率上具有独到的优势。但通过何种方式实现ETV2的过表达仍是值得考虑的问题。
中国专利CN 115515608A公开了一种利用腺病毒/慢病毒作为载体的方法,使ETV2基因转入小鼠体内,实现重编程,进而形成内皮细胞和血管。但该方法是通过体外改造腺病毒/慢病毒并使其转导目标细胞,将病毒所携带的核酸随机整合到宿主基因组上实现基因的过表达,其随机整合的特性会带来诸多不可预测的风险。同时,尽管病毒经过了体外改造,但是其生物学行为本身仍对人体有着潜在危害,故难以应用于临床细胞治疗。
发明内容
本发明的目的在于提供一种ETV2的ORF序列,能够高效表达ETV2蛋白,用于将干细胞分化为内皮细胞能够显著提升转化效率。
本发明提供的内皮细胞的制备方法,制备效率高、且临床应用风险较低。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种ETV2的ORF序列,所述ORF序列如SEQ ID NO.1所示。
本发明还提供了一种转录ETV2 mRNA的重组载体,所述重组载体包含初始载体和上述ORF序列。
优选的,所述重组载体还包含5’UTR调控序列和3’UTR调控序列;
所述5’UTR调控序列如SEQ ID NO.3所示;
所述3’UTR调控序列如SEQ ID NO.4所示。
优选的,所述初始载体为初始载体为质粒pUC57。
本发明还提供了上述ORF序列或上述重组载体在制备内皮细胞中的应用。
本发明还提供了一种过表达ETV2的方法,包括以下步骤:
将上述重组载体进行体外转录,得到ETV2 mRNA;
将ETV2 mRNA导入受体细胞,诱导表达,实现ETV2的过表达。
优选的,采用电转法将ETV2 mRNA导入受体细胞。
本发明还提供了一种内皮细胞的制备方法,包括以下步骤:
将上述ORF序列构建至初始载体中,得到重组载体;
将所得重组载体进行体外转录,得到ETV2 mRNA;
将ETV2 mRNA导入中胚层细胞,进行分化培养,得到内皮细胞。
本发明还提供了通过上述制备方法得到的内皮细胞。
本发明还提供了上述内皮细胞在制备血管生成促进剂中的应用。
本发明的有益效果:
本发明提供了一种ETV2的ORF序列,用于翻译能够得到更多的ETV2蛋白,表达效率更高。
本发明提供的内皮细胞制备方法采用了体外mRNA制备及mRNA转染技术,能够高效快速地实现内皮细胞的大量制备。制备方法中获得的ETV2 mRNA大小仅有1-2kb,导入中胚层细胞的效率接近100%。ETV2 mRNA导入中胚层细胞后表达时间较长,能够满足细胞命运重编程和内皮分化的需求。本发明提供的制备方法为内皮细胞直接注射到缺血部位、促进血管新生、改善组织灌注情况提供了稳定的细胞来源。
附图说明
图1为电转不同mRNA后24h ETV2蛋白条带图;
图2为免疫荧光检测ETV2表达情况的结果图;
图3为流式细胞检测结果图。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
设计并合成ETV2的人工ORF序列,结合密码子使用、GC含量,最小自由能等影响因素,以及去除其中的特定序列进行优化,优化后序列为:
ATGGACTTGTGGAATTGGGATGAAGCTTCTCCCCAGGAGGTGCCCCCCGGCAACAAGCTGGCCGGCCTGGAGGGCGCCAAGCTGGGCTTCTGCTTCCCCGATCTGGCCCTGCAGGGCGACACCCCTACCGCCACCGCCGAGACCTGCTGGAAGGGCACCAGCAGCAGCCTGGCCAGCTTCCCTCAGCTGGACTGGGGCTCTGCCCTGCTGCACCCTGAGGTGCCTTGGGGAGCCGAGCCCGACAGCCAGGCCCTGCCTTGGTCTGGCGACTGGACCGACATGGCCTGCACCGCCTGGGATAGCTGGTCTGGCGCCAGCCAGACACTGGGACCTGCCCCTCTGGGCCCTGGCCCTATTCCTGCTGCCGGAAGCGAAGGCGCCGCCGGCCAGAATTGTGTGCCTGTGGCCGGCGAGGCCACCAGCTGGTCTAGAGCCCAGGCCGCCGGCAGCAACACAAGCTGGGACTGTAGCGTGGGCCCTGACGGCGACACCTACTGGGGCAGCGGCCTGGGAGGCGAGCCTAGAACCGATTGTACCATCAGCTGGGGCGGCCCTGCCGGCCCTGACTGCACCACCAGCTGGAACCCTGGCCTGCACGCCGGCGGCACCACCAGCCTGAAGAGATACCAGAGCAGCGCCCTGACCGTGTGCAGCGAGCCCAGCCCCCAGAGCGACAGAGCCAGCCTGGCCAGATGCCCCAAGACCAACCACAGGGGCCCCATCCAGCTGTGGCAGTTCCTGCTGGAGCTGCTGCACGACGGCGCCAGGAGCAGCTGCATCAGATGGACCGGCAACAGCAGGGAGTTCCAGCTGTGCGACCCCAAGGAGGTGGCCAGGCTGTGGGGCGAGAGGAAGAGAAAGCCCGGCATGAACTACGAGAAGCTGAGCAGGGGCCTGAGGTACTACTACAGGAGGGACATCGTGAGCAAGAGCGGCGGCAGGAAGTACACCTACAGGTTCGGCGGCAGAGTGCCCAGCCTGGCCTACCCCGACTGCGCCGGCGGCGGCAGAGGCGCCGAGACCCAGTGA,如SEQ ID NO.1所示。
以及合成ETV2天然蛋白的ORF序列:
ATGGATTTGTGGAATTGGGATGAGGCATCACCTCAAGAGGTTCCGCCTGGTAACAAACTTGCAGGGCTCGAAGGAGCCAAATTAGGCTTCTGTTTCCCTGATCTGGCACTCCAAGGGGACACGCCGACAGCGACAGCAGAGACATGCTGGAAAGGTACAAGCTCATCCCTGGCAAGCTTCCCACAGCTGGACTGGGGCTCCGCGTTACTGCACCCAGAAGTTCCATGGGGGGCGGAGCCCGACTCTCAGGCCCTTCCGTGGTCCGGGGACTGGACAGACATGGCGTGCACAGCCTGGGACTCTTGGAGCGGCGCCTCGCAGACCCTGGGCCCCGCCCCTCTCGGCCCGGGCCCCATCCCCGCCGCCGGCTCCGAAGGCGCCGCGGGCCAGAACTGCGTCCCCGTGGCGGGAGAGGCCACCTCGTGGTCGCGCGCCCAGGCCGCCGGGAGCAACACCAGCTGGGACTGTTCTGTGGGGCCCGACGGCGATACCTACTGGGGCAGTGGCCTGGGCGGGGAGCCGCGCACGGACTGTACCATTTCGTGGGGCGGGCCCGCGGGCCCGGACTGTACCACCTCCTGGAACCCGGGGCTGCATGCGGGTGGCACCACCTCTTTGAAGCGGTACCAGAGCTCAGCTCTCACCGTTTGCTCCGAACCGAGCCCGCAGTCGGACCGTGCCAGTTTGGCTCGATGCCCCAAAACTAACCACCGAGGTCCCATTCAGCTGTGGCAGTTCCTCCTGGAGCTGCTCCACGACGGGGCGCGTAGCAGCTGCATCCGTTGGACTGGCAACAGCCGCGAGTTCCAGCTGTGCGACCCCAAAGAGGTGGCTCGGCTGTGGGGCGAGCGCAAGAGAAAGCCGGGCATGAATTACGAGAAGCTGAGCCGGGGCCTTCGCTACTACTATCGCCGCGACATCGTGCGCAAGAGCGGGGGGCGAAAGTACACGTACCGCTTCGGGGGCCGCGTGCCCAGCCTAGCCTATCCGGACTGTGCGGGAGGCGGACGGGGAGCAGAGACACAATGA,如SEQ ID NO.2所示。
与ETV2天然蛋白的ORF序列相比,本发明提供的ETV2人工ORF序列最小自由能更低,碱基分布更加合理。
通过常规分子克隆方法,基于基础质粒pUC57构建、扩增和提取含有上述两个ORF序列和其两侧上下游5’UTR、3’UTR调控序列的DNA质粒,能够编码ETV2的mRNA产物,UTR调控序列会影响mRNA的二级结构,影响翻译效率,采用本发明提供的调控序列能够实现最优效果。
5’UTR调控序列:
TAATACGACTCACTATAAGGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACC,如SEQ ID NO.3所示。
3’UTR调控序列:
GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA,如SEQ ID NO.4所示。
实施例2
分别将含有实施例1中ETV2的人工ORF序列和含有ETV2天然蛋白的ORF序列的DNA质粒线性化后,进行酶切,酶切体系如下:
BspQI-HF(NEB) 1μL、质粒DNA 10μg、10×CutSmart缓冲液(NEB) 2μL,无核酸酶水补至总体积20μL。
用涡旋混匀,离心机瞬离10s,37℃金属浴中反应3h。
质粒DNA沉淀:
(1)向反应后样品中加入0.7倍体积的异丙醇,充分混匀。
(2)13000rpm、室温离心15分钟使DNA沉淀。吸去上清后,加入1mL 70%乙醇洗沉淀,13000rpm、室温离心10分钟,去掉上清。
(3)去掉上清后,离心10s 将离心管璧的残留乙醇收集到管底,用吸头吸去残留的乙醇,室温放置2分钟吹干乙醇。加入50 µL超纯水溶解DNA沉淀。
(4)待沉淀完全溶解后,使用NanoDrop(Thermo Fisher)检测DNA浓度,置于-20℃冰箱保存。
分别得到含有实施例1中ETV2的人工ORF序列和含有ETV2天然蛋白的ORF序列的质粒。将获得的质粒DNA进行体外转录:
体外转录使用vazyme的体外转录试剂盒,货号:DD4202-01。帽子类似物使用vazyme的CAG Trimer(100mM),货号:DD4118-PC-00。
将10×IVT Buffer,100mM NTP以及CAG Trimer置于室温解冻,T7 Enzyme Mix 置于冰盒内。
取洁净的PCR管,将下列各组分依次加入管中(总:20μL):
10×Transcrption Buffer 2μL、100mM ATP 1.5μL、100mM CTP 1.5μL、100mMpUTP 1.5μL、100mM GTP 1.5μL、CAG Trimer(100mM) 1μL、DNA模板 1μg、T7 RNAPolymerase Mix 1μL,无核酸酶水补足至20μL。
移液枪轻柔混匀5s,离心机离心10s,置于恒温混匀仪中37℃孵育2.5h。
反应结束后加入1μL DNaseI,37℃孵育15min,去除线性化模板。
将mRNA进行纯化:
(1)将RNA Clean Beads平衡至室温,颠倒或旋涡振荡使磁珠充分混匀。
(2)加入36 μL RNA Clean Beads,加入IVT后的混合液中。
(3)用移液器吹打以充分混匀。
(4)室温孵育5 min,使RNA结合到磁珠上。
(5)将样品置于磁力架5min,待溶液澄清后,小心移除上清。
(6)保持样品始终处于磁力架中,加入200 μL新鲜配制的80%乙醇漂洗磁珠,室温孵育30s。
(7)移除上清,重复漂洗步骤,总计漂洗2次。
(8)保持样品始终处于磁力架中,开盖空气干燥磁珠10 min。
(9)将样品从磁力架上取出,加入适量体积的无核酸酶水,用移液器吹打10次以充分混匀,室温静置5 min。
(10)将样品置于磁力架5 min,待溶液澄清后,小心转移上清至一个新的离心管中。
使用NanoDrop检测mRNA浓度以及OD260/280的值,做好标记,置于-80℃冰箱保存,得到本发明的ETV2的ORF序列转录的mRNA、以及ETV2天然蛋白的ORF序列转录的mRNA。
实施例3
培养h-iPSC(人诱导多能干细胞)至细胞密度达到80%-90%时,进行传代;
传代24 h后,细胞密度约为30%时,更换培养基为中胚层细胞分化培养基,每隔24h进行一次换液,持续72 h,中胚层细胞分化培养基的配制方法如下表1所示:
表1 中胚层细胞分化培养基配制方法
试剂名称 | 终浓度 | 添加量 |
Advanced DMEM/F12 | - | 500 mL |
GlutaMAX (100×) | 1× | 5 mL |
Vc (30 mg/mL) | 60 μg/mL | 1 mL |
Pen/Strep (100×) | 0.2× | 1 mL |
CHIR99021 (20 mM) | 6 μM | 150 μL |
72 h后,分化所得细胞即为中胚层细胞(Mesoderm Progenitor Cells,MPCs)。
将所得的中胚层细胞用EDTA消化为单细胞悬液,经40 μm单细胞滤网过滤后计数,取1×106个细胞,用1×PBS洗涤一次后用100 μL mTeSR1重悬,并与1μg实施例2中得到的ETV2的人工ORF序列转录的mRNA、ETV2天然蛋白的ORF序列转录的mRNA分别混匀,加入到电转杯后,调用Lonza 电转仪(型号:Nucleofector 2B)中的程序B-016电转,完成电转后,将细胞加到基础培养基中培养,并铺在Matrigel包被好的培养板中继续分化96 h。每24 h按每孔2 mL更换基础培养基;基础培养基的配制方法如下表2所示:
表2 基础培养基配制方法
试剂名称 | 终浓度 | 添加量 |
Advanced DMEM/F12 | - | 500 mL |
GlutaMAX (100×) | 1× | 5 mL |
Vc (30 mg/mL) | 60 μg/mL | 1 mL |
Pen/Strep (100×) | 0.2× | 1 mL |
实验例1
按照实施例3中的哪种配方方法,分别将1μg、2μg实施例2中得到的ETV2的人工ORF序列转录的mRNA、ETV2天然蛋白的ORF序列转录的mRNA电转入细胞中(两组mRNA分别以两个浓度进行实验),24h后提取细胞蛋白进行Western Blot实验,经裂解蛋白、蛋白变性、凝胶配置、上样、电泳、转膜、漂洗、封闭、一抗孵育、二抗孵育与显色曝光后,结果如图1所示。
图1结果表明,在电转同样量的ETV2 mRNA的情况下,与ETV2天然蛋白的ORF序列相比,本发明提供的ETV2的人工ORF序列能够翻译成为更多的ETV2蛋白,对ETV2过表达具有重要意义。
实验例2
将实施例3中,完成电转并分化96h后的细胞铺在Matrigel包被的48孔板中。继续培养24 h后,弃去培养基上清,用200 μL 1×PBS洗涤一次,再用100 μL 4%多聚甲醛室温固定15 min。用200 μL 1×PBS洗涤一次后,加入100 μL 0.1% Triton X-100在室温使细胞膜通透10 min。用200 μL 1×PBS洗涤一次后,加入100 μL 2% 牛血清白蛋白(Bovine SerumAlbumin,BSA)室温封闭30 min。随后按1:300比例在2% BSA中稀释ETV2一抗,加入孔中室温染色30 min。用200 μL 1×PBS洗涤后按1:500比例在2% BSA中稀释Donkey anti-rabbitAlexa Fluor 488荧光二抗,加入孔中室温避光染色15 min。用200 μL 1×PBS洗涤后加入500 μL 1×4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)室温避光复染10 min。用200 μL 1×PBS洗涤后在荧光显微镜下观察,结果如图2所示。
如图2所示,可看到明显的ETV2表达结果,说明通过本发明成功实现了ETV2的高效表达。
实验例3
通过流式细胞技术鉴定内皮标志基因,将实施例3中,完成电转并分化96h后的细胞铺在Matrigel包被的6孔板中。继续培养48 h后,用TrypLE消化为单细胞悬液,用FACSbuffer洗涤一次,再用100 μL FACS buffer重悬,与1 μL CD31-APC和1 μL CD144-PE抗体混匀后,置于4℃避光染色15 min,用FACS buffer洗涤一次后重悬细胞,上机进行检测,结果如图3所示。
结果显示,超过90%的细胞为CD31+CD144+的内皮细胞,说明本发明能够简单高效地将h-iPSCs分化为内皮细胞。同时本发明所用的策略无需对干细胞进行基因编辑,提高了干细胞诱导分化过程中的安全性,具有广阔的临床应用前景。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种内皮细胞的制备方法,其特征在于,包括以下步骤:
将ETV2的ORF序列的核苷酸片段构建至初始载体中,得到重组载体;
将所得重组载体进行体外转录,得到ETV2 mRNA;
将ETV2 mRNA导入中胚层细胞,进行分化培养,得到内皮细胞;
所述ETV2的ORF序列的核苷酸片段如SEQ ID NO.1所示;
所述重组载体还包含5’UTR调控序列和3’UTR调控序列;
所述5’UTR调控序列如SEQ ID NO.3所示;
所述3’UTR调控序列如SEQ ID NO.4所示;
所述初始载体为质粒pUC57。
2.通过权利要求1所述的制备方法制备得到的内皮细胞。
3.权利要求2所述的内皮细胞在制备血管生成促进剂中的应用。
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