CN113999877B - 一种间充质干细胞药物的基因编辑方法及应用 - Google Patents
一种间充质干细胞药物的基因编辑方法及应用 Download PDFInfo
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Abstract
本发明公开了一种间充质干细胞药物的基因编辑方法及应用,本发明应用该方法制备出葡萄糖水平敏感的能够治疗糖尿病高血糖症的基因工程干细胞药物,操作简便,安全性高。本发明发现使用Cas9+血管内皮细胞分泌囊泡能将目的基因转染进入MSC内,高效地通过Crispr‑Cas9基因编辑系统实现精确编辑,增强其功能的同时保留MSC免疫调节和促生长特性的方法。
Description
技术领域
本发明属于生物医药技术领域,涉及一种细胞基因编辑技术,具体涉及一种间充质干细胞药物的基因编辑方法及应用。
背景技术
近年来干细胞治疗取得了猛进的发展,作为一种替代医学方案,效果已经得到了普遍认可。其中,间充质干细胞(MSCs)得到了广泛而深入的研究,国际细胞疗法学会(ISCT)给出了间充质干细胞的鉴定标准:1.贴壁生长;2.表面特异性抗原(Ag)表达:CD105,CD73,CD90阳性,而CD45,CD34,CD14或CD11b,CD79α,CD19和HLA-DR阴性;3.具有体外分化为成骨细胞,脂肪细胞和软骨细胞等三种细胞类型的能力。与其他干细胞相比,MSC具有许多特殊优势,例如,不存在伦理问题;易于从脂肪、骨髓和脐带中获取;一定的分化潜能(分化为脂肪细胞,成骨细胞,软骨细胞,成肌细胞等,良好的增殖率以良好的临床应用安全性。
MSC可以迁移到损伤部位,发挥免疫调节作用以及通过特异性分化修复损伤组织。因此,广泛用于实验和临床研究以及基因工程中,比如骨骼,心脏,软骨,中枢神经,皮肤等组织器官的再生修复,具有广阔的应用前景。然而,MSC本身的局限性也限制了其治疗功效。这些局限型包括:体内分化效率低,归巢率低,某些因子表达不足以及移植后细胞活力降低等。例如,在糖尿病的治疗中,MSC回输后几乎找不到MSC分化为β细胞的证据。然而,大量的实践证明,MSC本身具有一定的微环境稳态维持和修护能力,特别是能够抑制局部过激的免疫反应,保证自生的长期存活和防止β细胞的进一步衰竭,从而产生自身长期存活和改善生理微环境的双重效果。如果能特异性增强MSC某一方面的治疗功能并保持其自身免疫调节以及生长因子分泌的优势,是MSC治疗的一个巨大飞跃。
基因组编辑为此提供了解决方案。基因组编辑是目前基因治疗领域最热门的方法之一,使用可编程嵌合核酸酶(锌指核酸内切酶(ZFNs)、类转录激活因子效应物核酸酶(TALENs)和规律成簇间隔短回文重复序列相关系列(CRISPR/Cas))诱导DNA双链断裂,通过非同源端连接(nonhomologous end joining,NHEJ)或同源定向修复(homology-directedrepair,HDR)对细胞内DNA进行特异性序列修饰。相对于成本高昂的ZENs和TALENs(需特殊定制相应目标DNA对应的蛋白质-DNA结合域),CRISPR/Cas基于更简单的RNA引导DNA识别介导基因操纵,促进了低成本、高通量和多样化的遗传和表观遗传编辑,为探测基因功能和纠正突变提供了有力工具。同时,近年来,各种基因组编辑技术的出现为生物学家和研究人员提供了多种将序列特异性修饰引入细胞基因组的方法。包括:1.非病毒方法:物理方法(电动,显微注射,质粒注射,弹道注射)和化学方法(基于脂质体的方法,磷酸钙,DEAE葡聚糖,基于蛋白质的方法);2.病毒方法:RNA病毒(逆转录病毒,HIV(慢病毒)和DNA病毒(腺病毒,腺伴随病毒(AAV),单纯疱疹病毒)等。
利用基因编辑的方法对MSC进行改造,增强其治疗功能,并维持其免疫调节和促进集体修复的特性,有助于MSC细胞药物的研制开发。然而,间充质干细胞由于其自身特性,采用常规方法施行基因转染成功率较低,且衰老速度快,丧失了作为细胞药物的意义。如果发展出一种可以高效精准进行基因编辑,维持其干性状态、免疫调节能力和生长因子分泌能力的方法,对基于间充质干细胞的基因工程化细胞药物治疗发展有重要意义。
发明内容
发明目的:为了使MSC增强对特定疾病的治疗功能并维持其免疫调节和促生长特性,本发明提供一种MSC基因编辑方法可以高效的将目的基因转染进入MSC内,同时保留MSC免疫调节和促生长特性的方法从而获得了干细胞药物。
本发明还要解决的技术问题是提供了上述方法得到的干细胞药物。
本发明还要解决的技术问题是提供了上述方法或干细胞药物在制备特定疾病中的应用。
本发明还要解决的技术问题是提供了一种Cas9+EPC细胞株。
本发明尤其通过应用上述方法解决了一种将MSC改造为血糖水平响应,从而可以动态调节高血糖症的胰岛素分泌型MSC药物的方法,从而应用于血糖方面的药物的制备。
技术方案:为了解决上述技术问题,本发明提供了一种间充质干细胞药物基因编辑方法,所述基因编辑方法包括以下步骤:
1)制备表达Cas9工程化血管内皮细胞(Cas9+EPC细胞);
2)收集步骤1)制备的Cas9工程化血管内皮细胞分泌的工程化微囊泡(Cas9+MVs);
3)将引导靶向切割目标基因位点sgRNA和供体基因序列电转导入步骤2)获得的工程化微囊泡得到具有基因编辑功能的微囊囊泡;
4)将步骤3)得到具有基因编辑功能的微囊泡与间充质干细胞孵育即得干细胞药物。
其中,所述步骤1)的工程化血管内皮细胞的制备方法可以是:使用pLenti-CRISPR-V2、pMDLg/pRRE和pRSV-Rev慢病毒包装质粒转染HEK293细胞,收获病毒后,感染血管内皮细胞,随后用10μg/mL嘌呤霉素筛选3-5代,获得Cas9稳定表达的Cas9+EPC细胞株。
其中,所述步骤2)的收集工程化血管内皮细胞分泌的微囊泡的步骤为:收集血管内皮细胞或间充质干细胞或工程化血管内皮细胞的培养液上清,进行梯度离心去除细胞、细胞碎片、凋亡小体,将处理后的上清液通过超速离心,获得MVs沉淀,重悬MVs、超速离心、洗涤浓缩,重悬即得。
其中,所述步骤2)的工程化血管内皮细胞为稳定转染Cas9的血管内皮细胞。
其中,所述步骤3)中通过电转染的方式将引导靶向切割目标基因位点sgRNA和供体基因序列载入微囊泡中,所述转染条件为:电压1000~1200V,时间间隔10-15ms,2~5个脉冲。
其中,所述的基因编辑方法得到的干细胞药物。
本发明内容包括一种Cas9+EPC细胞株,所述Cas9+EPC细胞株的制备方法如下:使用pLenti-CRISPR-V2,pMDLg/pRRE和pRSV-Rev慢病毒包装质粒转染宿主细胞,收获病毒后,感染血管内皮细胞,随后用嘌呤霉素筛选3-5代,获得Cas9稳定表达的Cas9+EPC细胞株。
当所述的目的基因为血糖调节基因时,所述的sgRNA序列如SEQ ID NO:1所示,所述血糖调节质粒核苷酸序列如SEQ ID NO:2所示。
其中,所述的基因编辑方法或所述的干细胞药物在制备治疗糖尿病、高血糖症、糖尿病足或糖尿病并发症相关药物中的应用。
本发明内容可以包括Cas9稳定表达的血管内皮细胞株。
其中,所述的血管内皮细胞和间充质干细胞可以是最终受体的自体来源或异体来源。
其中,所述的血管内皮细胞和间充质干细胞可以来源于同一个体或不同个体。
本发明还包括一种基因编辑方法,所述方法仅通过更改供体目的基因序列的改变得到的其他功能的干细胞药物。
本发明通过一系列的试验对间充质干细胞的基因转染方法进行了测试,包括不同公司或自己制备的各种病毒载体、脂质体、高分子材料等,结果显示,间充质干细胞的转染成功率极低,偶尔有转染成功的试验,但间充质干细胞会快速衰亡。然而,我们意外的发现,如果使用血管内皮细胞外泌体作为转染材料,则能较好的解决上述问题。
有益效果:与现有技术相比,本发明具备以下优点:本发明经过长期的摸索,发现血管内皮细胞和间充质干细胞的外泌体能够较好的递送靶基因到间充质干细胞,并维持其活性。特别的,我们发现使用Cas9+血管内皮细胞分泌囊泡能将目的基因转染进入MSC内,高效地通过Crispr-Cas9基因编辑系统实现精确编辑,增强其功能的同时保留MSC免疫调节和促生长特性的方法。通过应用上述方法,本发明还提供了一种将MSC改造为能够响应血糖水平变化,可以动态调节高血糖症的胰岛素分泌型MSC药物的方法。
附图说明
图1使用电转、脂质体转染、病毒、293T细胞微囊泡转染间充质干细胞后的电镜图。
图2是干细胞细胞药物能够对不同浓度的葡萄糖做出不同的响应,胰岛素的释放量随血糖浓度不同而变化,而对照细胞由不能够对培养基中的血糖浓度变化做出响应,升高或降低胰岛素的合成水平。
具体实施方式
以下结合实施例对本发明作进一步说明,实施例是用于说明本发明,而不是用于限制本发明的范围。本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明做出各种修改和改变,以使其使用各种用途和条件。
实施例中未注明具体条件者,皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。除特殊注明外,本发明所采用的均为该领域现有技术。
sgRNA寡核苷酸序列为:
血糖调节质粒寡核苷酸序列为:
以上序列是由生工生物工程(上海)股份有限公司合成。
实施例1
为比较电转、脂质体、病毒和293T细胞产生的微囊泡(293T-MVs)、血管内皮细胞微囊泡(EPC-MVs)和间充质干细胞微囊泡(MSC-MVs)为载体递送目标基因至脂肪间充质干细胞(间充质干细胞,赛业(广州)生物科技有限公司)的效果,进行了如下试验:
1)电转:利用Neon电转染系统将商品化的pEF-GFP质粒(上海柯雷生物科技有限公司)载入间充质干细胞中,转染条件为:当电压1000V,时间间隔10ms,2个脉冲;
2)脂质体转染:将商品化的pEF-GFP质粒(上海柯雷生物科技有限公司)通过电转方式载入脂质体,并通过Lipofections 2000(美国赛默飞公司)转染进入间充质干细胞;
3)病毒转染:使用pLenti-EGFP-V2,pMDLg/pRRE和pRSV-Rev(Addgene)慢病毒包装质粒转染293T细胞,并收集病毒。病毒与间充质干细胞共同孵育72小时,递送EGFP基因至间充质干细胞;
4)293T细胞微囊泡转染:收集293T微囊泡,将商品化的pEF-GFP质粒通过电转方式载入293T-MVs,将电转负载pEF-GFP质粒的293T-MVs与间充质干细胞孵育72小时,递送EGFP基因至间充质干细胞;
5)血管内皮细胞微囊泡转染:收集血管内皮细胞微囊泡,将商品化的pEF-GFP(上海柯雷生物科技有限公司)质粒通过电转方式载入EPC-MVs,将电转负载EGFP质粒的血管内皮细胞微囊泡与间充质干细胞孵育72小时,递送EGFP基因至间充质干细胞;
6)间充质干细胞微囊泡转染:收集间充质干细胞微囊泡,将商品化的pEF-GFP质粒通过电转方式载入MSC-MVs,将电转负载EGFP质粒的间充质干细胞微囊泡与间充质干细胞孵育72小时,递送EGFP基因至间充质干细胞;
图1显示上述六种方式中,使用电转、脂质体转染、病毒、293T细胞微囊泡转染后,间充质干细胞均快速凋亡,而使用血管内皮细胞微囊泡和间充质干细胞微囊泡则获得了比较的转染效果,说明血管内皮细胞微囊泡和间充质干细胞微囊适用于转染间充质干细胞,毒性小,效率高。
实施例2
制备工程化血管内皮细胞:使用pLenti-CRISPR-V2,pMDLg/pRRE和pRSV-Rev(Addgene公司购买)慢病毒包装质粒转染HEK293细胞,收获病毒后,感染血管内皮细胞,随后用10μg/mL嘌呤霉素筛选5代,获得Cas9稳定表达的Cas9+EPC细胞株;
收集Cas9+血管内皮细胞分泌的工程化微囊泡:收集Cas9+EPC细胞株的培养液上清,进行梯度离心(600g,30min去除细胞;2000g,20min去除细胞碎片;4500g,30min去除凋亡小体)。将处理后的上清液通过L-80XP Ti-70转子(Beckman Coulter,美国)以20,000g的速度超速离心45min,获得MVs沉淀。随后,使用25mL 0.22μm滤过的磷酸盐缓冲液(DPBS)重悬MVs,并在20,000g转速下超速离心1h,对获得的MVs进一步洗涤浓缩,最后用100μL 1 xDPBS重悬,存放于-80℃超低温冰箱。所有的离心步骤均在4℃进行。
负载:利用Neon电转染系统将人工合成的sgRNA(SEQ ID NO:1)和人工合成的目标DNA质粒(SEQ ID NO:2)载入Cas9+血管内皮细胞分泌的工程化微囊泡中,转染条件为:当电压1000V,时间间隔10ms,2个脉冲,制备得到sgRNA-CAS微囊泡。
转染:将106个sgRNA-CAS微囊泡与105个间充质干细胞孵育72小时得到干细胞药物。
实施例3葡萄糖冲击试验
为了验证该干细胞药物的功能,我们进行了葡萄糖冲击试验。方法如下:将106个干细胞药物与106对照细胞(293T细胞)接种到6孔板中。培养1周后,洗涤细胞,并在添加低葡萄糖(3.3mm D-葡萄糖)的10%FBS/DMEM中孵育3h,然后与含有0.1%BSA的DMEM培养基孵育1h。最后,将培养基分别改变为含有0.1%BSA的含有0mM/L,5mM/L,15mM/L或25mM葡萄糖(高葡萄糖)的DMEM培养基,孵育1.5小时,然后收集上清液,并通过Elisa测定释放的胰岛素的量。如图2所述,本发明的干细胞药物能够对不同浓度的葡萄糖做出不同的响应,胰岛素的释放量随血糖浓度不同而变化。而对照细胞由不能够对培养基中的血糖浓度变化做出响应,升高或降低胰岛素的合成水平,表明本发明对于干细胞编辑制备干细胞药物有重要意义。
序列表
<110> 东南大学
<120> 一种间充质干细胞药物的基因编辑方法及应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> sgRNA寡核苷酸序列(Artificial Sequence)
<400> 1
gagagagacc ctcactgctg 20
<210> 2
<211> 1317
<212> DNA
<213> 血糖调节质粒寡核苷酸序列(Artificial Sequence)
<400> 2
cgccgaactc agaggccggc cccagaaaac ccgagcgagt agggggcggc gcgcaggagg 60
gaggagaact gggggcgcgg gaggctggtg ggtgtggggg gtggagatgt agaagatgtg 120
acgccgcggc ccggcgggtg ccagattagc ggacgcggtg cccgcggttg caacgggatc 180
ccgggcgctg cagcttggga ggcggctctc cccaggcggc gtccgcggag acacccatcc 240
gtgaacccca ggtcccgggc cgccggctcg ccgcgcacca ggggccggcg gacagaagag 300
cggccgagcg gctcgaggat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 360
gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 420
cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 480
acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 540
gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 600
atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 660
ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 720
cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 780
ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 840
ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 900
cgcaagcccg gtgcctgagg atccggcgca acaaacttct ctctgctgaa acaagccgga 960
gatgtcgaag agaatcctgg accgatggcc ctgtggatgc gcctcctgcc cctgctggcg 1020
ctgctggccc tctggggacc tgacccagcc gcagcctttg tgaaccaaca cctgtgcggc 1080
tcacacctgg tggaagctct ctacctagtg tgcggggaac gaggcttctt ctacacaccc 1140
aggaccaagc gggaggcaga ggacctgcag gtggggcagg tggagctggg cgggggccct 1200
ggtgcaggca gcctgcagcc cttggccctg gagggatccc ggcagaagcg tggcattgtg 1260
gaacaatgct gtaccagcat ctgctccctc taccagctgg agaactactg caactag 1317
Claims (6)
1.一种间充质干细胞药物的基因编辑方法,其特征在于,所述基因编辑方法包括以下步骤:
1)制备表达Cas9工程化血管内皮细胞;
2)收集步骤1)制备的Cas9工程化血管内皮细胞分泌的工程化微囊泡;
3)将引导靶向切割目标基因位点sgRNA和供体基因序列导入步骤2)获得的工程化微囊泡得到具有基因编辑功能的微囊泡;
4)将步骤3)得到具有基因编辑功能的微囊泡与间充质干细胞孵育即得干细胞药物;
所述sgRNA的序列如SEQ ID NO:1所示,所述供体基因序列如SEQ ID NO:2所示。
2.根据权利要求1所述的间充质干细胞药物的基因编辑方法,其特征在于,所述步骤1)的Cas9工程化血管内皮细胞的制备方法包括:使用pLenti-CRISPR-V2,pMDLg/pRRE和pRSV-Rev慢病毒包装质粒转染宿主细胞,收获病毒后,感染血管内皮细胞,随后用嘌呤霉素筛选3-5代,获得Cas9稳定表达的Cas9+EPC细胞株。
3.根据权利要求1所述的间充质干细胞药物的基因编辑方法,其特征在于,所述步骤2)的收集Cas9工程化血管内皮细胞分泌的工程化微囊泡的步骤为:收集Cas9工程化血管内皮细胞的培养液上清,进行梯度离心去除细胞、细胞碎片、凋亡小体,将处理后的上清液通过超速离心,获得MVs沉淀,重悬MVs、超速离心、洗涤浓缩,重悬即得。
4.根据权利要求1~3任一项所述的间充质干细胞药物的基因编辑方法,其特征在于,所述步骤3)中通过电转染的方式将引导靶向切割目标基因位点sgRNA和供体基因序列载入微囊泡中,所述转染条件为:电压1000~1200V,时间间隔10-15ms,2-5个脉冲。
5.权利要求1~4任一项所述的基因编辑方法得到的干细胞药物。
6.权利要求1~4任一项所述的基因编辑方法或权利要求5所述的干细胞药物在制备治疗糖尿病、高血糖症、糖尿病足相关药物中的应用。
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