CN112920989B - 一种肝脏类器官模型及其建立方法、用途以及治疗肝细胞铁死亡的药物组合物 - Google Patents
一种肝脏类器官模型及其建立方法、用途以及治疗肝细胞铁死亡的药物组合物 Download PDFInfo
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Abstract
本发明涉及医药技术领域,具体地,本发明涉及一种肝脏类器官模型及其建立方法、用途以及治疗肝细胞铁死亡的药物组合物。本发明提供一种2D肝样细胞铁过载模型和3D肝脏类器官铁过载模型,依靠2D肝样细胞分化技术和3D肝类器官培养技术,建立强大可靠的体外肝脏疾病模型,并基于该疾病模型筛选用于缓解和/或治疗肝细胞铁过载或铁死亡的药物。特别是3D肝脏类器官更接近于生理状态的肝组织,对疾病模型和药物筛选有很好的应用前景。
Description
技术领域
本发明涉及医药技术领域,具体地,本发明涉及一种肝脏类器官模型及其建立方法、用途以及治疗肝细胞铁死亡的药物组合物。
背景技术
线粒体DNA缺失综合征(MDS,Mitochondrial DNA depletion syndrome)是一类常染色体隐性遗传病,由于维持线粒体DNA合成的核基因发生突变,线粒体DNA无法正常合成,线粒体DNA拷贝数严重减少,导致多种组织器官功能障碍的严重疾病,受累器官通常有肝脏、脑、肾和肌肉等。目前已知有9种基因突变会导致MDS,DGUOK是其中之一。DGUOK突变的MDS患者通常在出生后6个月内发病,多数在发病后一年内死亡。肝组织活检显示肝脏铁沉积、肝脏脂肪变性、胆汁淤积、肝小叶结构崩解等。患者预后极差,通常死于严重的肝衰竭。由于这类疾病病情复杂、发病机理不清、且尚无针对病因的特效药物,目前的治疗手段仅有药物治疗缓解些许症状或采取肝移植方案。
类器官(organoid)培养是一种体外三维培养的精准治疗的前沿技术,建立起一个模拟人体内环境的模型,患者细胞形成类似器官的组织结构,很好地保留了细胞与细胞、细胞与细胞外基质之间的作用情况。因此相对于传统细胞系,该模型具有更高的临床相关性和个体多样性,更加适合建立类器官标本库并用于评价化合物的药效。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此,本发明的一个目的在于提供一种2D肝样细胞铁过载模型和3D肝脏类器官铁过载模型,并基于该疾病模型筛选用于缓解和/或治疗肝细胞铁过载或铁死亡的药物。其中,本发明中的2D肝样细胞和3D肝脏类器官通过iPS细胞分化获得,所述iPS细胞含有与线粒体DNA缺失综合征相关的基因突变。3D肝脏类器官更接近于生理状态的肝组织,对疾病模型和药物筛选有很好的应用前景。
为此,本发明一方面提供一种2D肝样细胞铁过载模型的建立方法。根据本发明的实施例,所述建立方法包括:
(1)利用iPS细胞分化获得2D肝样细胞;
(2)利用柠檬酸铁铵与所述2D肝样细胞接触,以便获得2D肝样细胞铁过载模型,其中,所述iPS细胞源自哺乳动物的体细胞;
所述iPS细胞中含有与线粒体DNA缺失综合征相关的基因发生的突变,
所述柠檬酸铁铵的浓度为5-10mM。
目前世界上对DGUOK突变MDS的研究并不多,在临床病例型研究中报道了绝大多数DGUOK突变的患者实验室检查发现存在肝脏铁沉积,血清学检查显示血清铁蛋白和转铁蛋白升高。肝脏作为人体内主要的铁贮器官,铁过载时肝脏首当其冲成为铁毒性攻击的主要部位。肝脏中过多的铁可导致氧化性损伤、肝硬化、肝细胞癌,并最终导致肝衰竭。铁的氧化还原活性可以产生过多的自由基导致脂质过氧化,启动细胞死亡,这一过程称为铁死亡。
对于MDS这类遗传性疾病,发明人利用iPS技术构建疾病模型,可以获得具有肝细胞分化能力的iPS细胞,并结合CRISPR/cas9基因编辑技术对突变基因进行修复,保证遗传背景的一致性。利用具有基因突变引起的MDS的哺乳动物(例如人)的体细胞,获取iPS细胞,其中iPS细胞中含有线粒体DNA缺失综合征相关的基因突变,利用iPS细胞分化获得2D肝样细胞,通过特定浓度的柠檬酸铁铵与该2D肝样细胞接触,能够获得2D肝样细胞铁过载模型,该铁过载模型与患有MDS患者的肝脏中肝细胞铁沉积类似,因此,利用该2D肝样细胞铁过载模型可用于筛选缓解和/或治疗肝细胞铁过载或铁死亡的药物。根据本发明的实施例,所述与线粒体DNA缺失综合征相关的基因选自DGUOK、POLG、TK2、TYMP、MPV17、SUCLA2、SUCLG1、RRM2B、C10orf2中的至少之一。
根据本发明的实施例,所述柠檬酸铁铵的浓度为5mM。
根据本发明的实施例,所述iPS细胞通过以下方法获取:
1)将含有OCT4、SOX2、KLF4、c-MYC基因的病毒感染哺乳动物的体细胞,
2)对感染后第六天的体细胞进行分盘:将细胞用0.25%胰酶消化传代,按照每盘30-40×104个细胞/6cm盘的密度接种细胞,用4mL再编程培养基2进行培养,每天更换培养基,感染后16天将所述再编程培养基2更换为mTeSR培养基继续培养,其中,接种细胞所用的盘为用Matrigel包被的盘;
3)根据培养获得的克隆的形态挑取iPS细胞克隆。
根据本发明的实施例,所述再编程培养基2包括500mL F12培养基、10ml ITS、32mg维生素c、2.5g NaCl、5ugbFGF以及100uM丁酸钠。
根据本发明的实施例,所述哺乳动物的体细胞为皮肤成纤维细胞。
本发明第二方面提供一种利用第一方面所述的建立方法获得的2D肝样细胞铁过载模型。
本发明第三方面提供一种3D肝脏类器官铁过载模型的建立方法。根据本发明的实施例,所述建立方法包括:
1)对第一方面所述的建立方法获得的2D肝样细胞进行3D肝脏类器官培养,以便获得3D肝脏类器官;
2)利用柠檬酸铁铵与所述3D肝脏类器官接触,以便获得3D肝脏类器官铁过载模型,
其中,所述柠檬酸铁铵的浓度为5-10mM。
因为肝脏是多细胞构成的一个结构和功能复杂的立体器官,单纯以2D肝样细胞分化为模型不足以满足对疾病的深入研究,通过3D肝类器官培养技术,可以获得包含肝细胞、胆管细胞、星状细胞等多种细胞的肝类器官,这种肝类器官具有三维组织结构、肝功能更成熟的巨大优点,更接近于生理状态的肝组织,对疾病模型和药物筛选有很好的应用前景。
利用具有基因突变引起的MDS的哺乳动物(例如人)的体细胞,获取iPS细胞,其中iPS细胞中含有线粒体DNA缺失综合征相关的基因突变,利用iPS细胞分化获得3D肝脏类器官,通过特定浓度的柠檬酸铁铵与该3D肝脏类器官接触,能够获得3D肝脏类器官铁过载模型,该铁过载模型与患有MDS患者的肝脏中肝细胞铁沉积类似,因此,利用该3D肝脏类器官铁过载模型可用于筛选缓解和/或治疗肝细胞铁过载或铁死亡的药物。
根据本发明的实施例,进行所述3D肝脏类器官培养的方法包括:
(Ⅰ)将所述2D肝样细胞用Accutase消化10-15min,加入DMEM/F12培养基重悬,弃上清;
(Ⅱ)用Matrigel重悬细胞,接种到培养板中,置于细胞培养箱培养20-30min,待Matrigel凝固,加入肝类器官培养基,每3天换液一次;
(Ⅲ)在培养箱中培养14天后,按1:4比例进行传代,每3天换液一次,以便获得3D肝脏类器官;
所述肝类器官培养基组成:
AdDMEM/F12、0.5%青霉素-链霉素、1%GlutaMAX、10mM HEPES、1%B27 minusvitamin A、15%R-spodin1条件培养基、3μM ChIR99021、10mM烟酰胺、10nM胃泌素、50ngmL-1EGF、20ng mL-1TGF-α、100ng mL-1FGF7、100ng mL-1FGF-10、50ngmL-1HGF、2mM A83-01、10μM Y-27632、1μM地塞米松,10ng mL-1OncostatinM。
本发明第四方面提供第三方面所述的建立方法获得的3D肝脏类器官铁过载模型。
本发明第五方面提供第一方面所述的建立方法获得的2D肝样细胞铁过载模型、第二方面所述的2D肝样细胞铁过载模型、第三方面所述的建立方法获得的3D肝脏类器官铁过载模型、第四方面所述的3D肝脏类器官铁过载模型在筛选药物中的用途。根据本发明的实施例,所述药物用于缓解和/或治疗肝细胞铁过载或铁死亡。
本发明第六方面提供一种筛选缓解和/或治疗肝细胞铁过载或铁死亡的药物的方法。根据本发明的实施例,所述方法包括:
将第一方面所述的建立方法获得的2D肝样细胞铁过载模型、第二方面所述的2D肝样细胞铁过载模型、第三方面所述的建立方法获得的3D肝脏类器官铁过载模型、第四方面所述的3D肝脏类器官铁过载模型中的至少之一与待测药物接触,所述2D肝样细胞和/或3D肝脏类器官中细胞内的游离铁含量降低、细胞还原型GSH含量升高、细胞中铁蛋白和溶酶体共定位降低、细胞脂质ROS降低中的至少之一,是所述待测药物能够缓解和/或治疗肝细胞铁过载或铁死亡的指示。通过iPS技术结合CRISPR/cas9基因修复技术获得MDS病人来源iPS细胞,通过2D肝样细胞分化和3D肝类器官培养,建立稳定可靠的体外肝脏疾病模型,揭示铁在MDS病人肝衰竭中的致病机制,并在此基础上筛选出化合物去铁胺(DFO)、Ferrostatin-1(Fer-1)、N-乙酰半胱氨酸(NAC)可以缓解DGUOK突变的MDS病人肝细胞铁过载导致的铁死亡。
本发明第七方面提供去铁胺、Ferrostatin-1、N-乙酰半胱氨酸在制备缓解和/或治疗肝细胞铁过载或铁死亡的药物中的用途。根据本发明的实施例,所述肝细胞铁过载或铁死亡由与线粒体DNA缺失综合征相关的基因突变引起。
根据本发明的实施例,所述与线粒体DNA缺失综合征相关的基因选自DGUOK、POLG、TK2、TYMP、MPV17、SUCLA2、SUCLG1、RRM2B、C10orf2中的至少之一。
发明人基于上述筛选药物的方法,筛选获得去铁胺、Ferrostatin-1、N-乙酰半胱氨酸,发现这三种药物能够缓解和/或治疗肝细胞铁过载或铁死亡,其中肝细胞铁过载或铁死亡由与线粒体DNA缺失综合征相关的基因突变引起。
本发明第八方面提供一种用于缓解和/或治疗肝细胞铁过载或铁死亡的药物组合物。根据本发明的实施例,所述药物组合物中含有去铁胺、Ferrostatin-1、N-乙酰半胱氨酸中的至少之一,其中,所述肝细胞铁过载或铁死亡由与线粒体DNA缺失综合征相关的基因突变引起;任选地,所述与线粒体DNA缺失综合征相关的基因选自DGUOK、POLG、TK2、TYMP、MPV17、SUCLA2、SUCLG1、RRM2B、C10orf2中的至少之一。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了本发明利用离体的患者皮肤的成纤维细胞通过iPS诱导,获得肝样细胞和肝类器官,并基于细胞和类器官进行药物筛选的过程;
图2显示了利用实施例1中方法获得的由离体的患者皮肤成纤维细胞诱导成iPS细胞的显微图片;
图3A显示了实施例2中对源自两个病人的iPS样本进行CRISPR/cas9基因修复技术的设计;
图3B显示了测序结果显示实施例2中经过CRISPR/cas9基因修复技术病人1和病人2的DGUOK基因突变得到了修复;
图4显示了实施例3中患者iPS细胞诱导分化成2D肝样细胞过程各阶段的细胞状态显微图片以及肝细胞maker的鉴定(图中白色箭头所指为肝细胞maker表达区域);
图5显示了实施例4中患者3D肝类器官肝细胞maker的鉴定;
图6A显示了实施例5中用不同浓度FAC(柠檬酸铁铵)处理正常人、病人和病人修复肝类器官细胞死亡的检测和统计结果;
图6B显示了实施例5中用不同浓度FAC处理正常人、病人和病人修复肝样细胞细胞存活率的统计结果;
图7A显示了实施例6中正常人、病人和病人修复的肝样细胞GSH含量的检测结果;
图7B显示了实施例6中正常人、病人和病人修复的肝样细胞FAC处理前后游离铁含量的检测结果
图7C显示了实施例6中正常人、病人和病人修复的肝样细胞铁蛋白和溶酶体共定位的免疫荧光图;
图8A显示了实施例7中分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝样细胞的脂质ROS检测的结果,其中DMSO组为对照;
图8B显示了实施例7中分别用FAC+DMSO、FAC+NAC处理的正常人、病人和病人修复的肝样细胞的脂质ROS检测的结果,其中DMSO组为对照;
图8C显示了实施例7中分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝样细胞存活率的结果,其中DMSO组为对照;
图8D显示了实施例7中分别用FAC+DMSO、FAC+NAC处理的正常人、病人和病人修复的肝样细胞存活率的结果,其中DMSO组为对照;
图8E显示了实施例7中分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝类器官细胞死亡的结果,其中DMSO组为对照;
图8F显示了实施例7中分别用FAC、FAC+NAC处理的正常人、病人和病人修复的肝类器官细胞死亡的结果。
具体实施方式
根据本发明的具体实施例,本发明提供一种2D肝样细胞铁过载模型的建立方法,所述建立方法包括:
(1)利用iPS细胞分化获得2D肝样细胞;
(2)利用柠檬酸铁铵与所述2D肝样细胞接触,以便获得2D肝样细胞铁过载模型,其中,所述iPS细胞源自哺乳动物的体细胞;
所述iPS细胞中含有与线粒体DNA缺失综合征相关的基因发生的突变,
所述柠檬酸铁铵的浓度为5-10mM,所述与线粒体DNA缺失综合征相关的基因选自DGUOK、POLG、TK2、TYMP、MPV17、SUCLA2、SUCLG1、RRM2B、C10orf2中的至少之一,优选为DGUOK基因。
根据本发明优选的实施例,所述柠檬酸铁铵的浓度为5mM。
根据本发明的具体实施例,所述iPS细胞通过以下方法获取:
1)将含有OCT4、SOX2、KLF4、c-MYC基因的病毒感染哺乳动物的体细胞,
2)对感染后第六天的体细胞进行分盘:将细胞用0.25%胰酶消化传代,按照每盘30-40×104个细胞/6cm盘的密度接种细胞,用4mL再编程培养基2进行培养,每天更换培养基,感染后16天将所述再编程培养基2更换为mTeSR培养基继续培养,其中,接种细胞所用的盘为用Matrigel包被的盘;
3)根据培养获得的克隆的形态挑取iPS细胞克隆;
所述再编程培养基2包括500mL F12培养基、10ml ITS、32mg维生素c、2.5g NaCl、5ugbFGF以及100uM丁酸钠。
根据本发明的具体实施例,所述哺乳动物的体细胞为皮肤成纤维细胞,例如,患有MDS病人的皮肤成纤维细胞。
根据本发明的具体实施例,本发明提供一种3D肝脏类器官铁过载模型的建立方法,所述建立方法包括:
1)对2D肝样细胞进行3D肝脏类器官培养,以便获得3D肝脏类器官;
2)利用柠檬酸铁铵与所述3D肝脏类器官接触,以便获得3D肝脏类器官铁过载模型,
其中,所述柠檬酸铁铵的浓度为5-10mM。
进行所述3D肝脏类器官培养的方法包括:
(Ⅰ)将所述2D肝样细胞用Accutase消化10-15min,加入DMEM/F12培养基重悬,弃上清;
(Ⅱ)用Matrigel重悬细胞,接种到培养板中,置于细胞培养箱培养20-30min,待Matrigel凝固,加入肝类器官培养基,每3天换液一次;
(Ⅲ)在培养箱中培养14天后,按1:4比例进行传代,每3天换液一次,以便获得3D肝脏类器官;
所述肝类器官培养基组成:
AdDMEM/F12、0.5%青霉素-链霉素、1%GlutaMAX、10mM HEPES、1%B27 minusvitamin A、15%R-spodin1条件培养基、3μM ChIR99021、10mM烟酰胺、10nM胃泌素、50ngmL-1EGF、20ng mL-1TGF-α、100ng mL-1FGF7、100ng mL-1FGF-10、50ngmL-1HGF、2mM A83-01、10μM Y-27632、1μM地塞米松,10ng mL-1OncostatinM。
图1显示了本发明利用离体的患者皮肤的成纤维细胞通过iPS诱导,获得肝样细胞和肝类器官,并基于细胞和类器官进行药物筛选的过程。利用该筛选方法能够获得缓解和/或治疗肝细胞铁过载或铁死亡的药物。
下面详细描述本发明的实施例。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1iPS细胞诱导
将患有线粒体DNA缺失综合征的患者的成纤维细胞诱导成iPS细胞,诱导过程如下:
DGUOK突变病人iPS细胞的诱导包括病毒包装、病毒感染、细胞分盘以及挑取克隆,实验步骤如下:
1、病毒包装:293T细胞按照400×104个细胞/10cm盘的细胞密度接种,待293T细胞的密度约为80%时为最适转染密度,用磷酸钙转染方法转染pMXs-hOCT4、pMXs-hSOX2、pMXs-hKLF4、pMXs-hc-MYC四种质粒(购自addgene,质粒编号依次为#17964、#17218、#17219、#17220),置于5%CO2、37℃培养箱过夜。第二天上午更换9mL新鲜293T培养基。收集含有病毒的293T细胞的培养基,用0.45μm滤膜过滤,加入polybrene(终浓度为8μg/mL),混匀。每盘293T细胞再加入9mL新鲜293T培养基,用于第二次的病毒收集。
2、病毒感染:将六孔板里的培养DGUOK突变病人的成纤维细胞的培养基替换为2mL新鲜的成纤维细胞培养基,然后加入含有OCT4、SOX2、KLF4、c-MYC基因的四种病毒上清(六孔板每孔每种病毒各2mL),每个孔内培养基的总体积为10mL,感染过夜后换新鲜成纤维细胞培养基。第二天进行第二次病毒感染。第三天,更换DFBS培养基。
3、细胞分盘:第二次感染后第六天后进行分盘:提前2小时用2mL Matrigel包被6cm盘,将细胞用0.25%胰酶消化并传代,按照每盘40×104个细胞/6cm盘的密度接种细胞,用4mL再编程培养基2(Reprogramming medium 2)进行培养。接下来的几天每天更换再编程培养基2。感染后16天换成mTeSR培养基继续培养。
再编程培养基2包括500mL F12培养基、10ml ITS、32mg维生素c、2.5g NaCl、5ugbFGF以及100uM丁酸钠。
4、挑取克隆(根据克隆的形态决定挑克隆日期,避免克隆分化)。
通过以上方法获得由患者的成纤维细胞诱导形成的iPS细胞,患者的成纤维细胞以及iPS细胞的形态如附图2。
实施例2对患者iPS细胞通过CRISPR/cas9技术进行修复
将两个病人的iPS细胞通过CRISPR/cas9技术进行修复,得到了DGUOK突变位点修复的iPS细胞,图3A显示了对这两位病人的iPS样本进行CRISPR/cas9基因修复技术的设计过程。
CRISPR/cas9基因修复技术具体步骤如下:
1、CRISPR/cas9切割靶位点的设计
利用CRISPR设计网站(http:///crispr.mit.edu/)进行CRISPR/cas9切割靶位点的设计,根据网站的打分,选择得分较高的Guide序列,选择好sgRNA后,需要加上酶切位点,并设计出sgRNA的反向互补配对链。将设计好的2对sgRNA对应的DNA序列送公司合成。
病人1的sgRNA1对应的DNA序列:5’-CACCGTCTGATGAACATTCCAGTGC-3’,如SEQ IDNO:1所示;
病人1的sgRNA2对应的DNA序列:5’-AAACGCACTGGAATGTTCATCAGAC-3’,如SEQ IDNO:2所示;
病人2的sgRNA1对应的DNA序列:5’-CACCGCCTTCGATGGAGAGCCTTCG-3’,如SEQ IDNO:3所示;
病人2的sgRNA2对应的DNA序列:5’-AAACCGAAGGCTCTCCATCGAAGGC-3’,如SEQ IDNO:4所示。
2、CRISPR/cas9质粒的构建
1)将上述合成好的sgRNA的反向互补配对链进行退火,得到双链sgRNA;
2)将pX330-tomato酶切回收(pX330购自Addgene,tomato为荧光标记);
3)连接:将退火形成的双链DNA与和酶切线性化的pX330-tomato连接形成特异性打靶质粒,16℃连接4h;
4)转化:将上述连接产物转化DH5α感受态,在LB固体培养板上(含50ug/mL氨苄青霉素)置于37℃温箱中培养过夜。第二天观察LB平板上长出的菌落。挑取单克隆菌落,放入3mL LB液体培养基(含氨苄青霉素)中,37℃摇床上摇菌过夜;
5)质粒小提和鉴定:菌液用天根质粒小提试剂盒提取质粒,并送测序:
6)质粒去内毒素提取:在50mL离心管中加入15mL的LB培养基(含氨苄),吸取适量序列正确的菌液,加入培养基中,37℃摇床中培养12-14小时,使用Omega去内毒素质粒微量提取试剂盒提取质粒。
3、修复模版的构建
使用更为方便高效的单链供体寡核苷酸(single-stranded donoroligonucleotides,ssODN)作为donor。ssODN总长度不超过150nt,长度跨越突变位点和CRISPR/cas9切割位点,考虑CRISPR/cas9可能重复切割的问题,于是在ssODN对应的CRISPR/cas9识别序列处根据密码子偏好原则设计了同义突变。
4、质粒和模板的电转
利用电转化法将CRISPR/cas9质粒和修复模版导入实施例1中获得的病人iPS细胞,当病人iPS细胞长到密度大约80%左右时实施电转实验。电转完成后使用mTeSR(含Y-27632)培养基,置于37℃培养箱过夜。第二天观察细胞,换液,培养基为mTeSR(含Y-27632),第三天换液时撤去Y-27632。
5、修复克隆的筛选和鉴定
1)待iPS克隆长至肉眼可见比较大且克隆之间并未连接时即可挑取单克隆;
2)突变修复鉴定:将所有克隆进行PCR,送测序鉴定突变是否被修复。
附图3B显示通过CRISPR/cas9基因修复技术,病人1和病人2的iPS细胞中DGUOK基因突变被成功修复。
实施例32D肝样细胞分化
将iPS细胞分化为2D肝样细胞步骤如下:
体外肝分化采用无血清分化方案,经过四个阶段21天分化,方法如下:
1)传代前一天用新鲜Matrigel提前包被培养板,37℃培养箱过夜。当实施例1中获得的病人iPS细胞长到80%密度时,使用Accutase进行传代,传代时要加Y-27632。
2)传代第二天iPS细胞长至90%,不要超过24h,开始换肝分化培养基(培养基配方参考文献S.Li,J.Guo,Z.Ying,S.Chen,L.Yang,K.Chen,Q.Long,D.Qin,D.Pei and X.Liu,Hepatology.2015,61,5)进行肝分化,分为4个阶段,共21天,包括:定向内胚层分化、肝前体细胞分化、肝前体细胞扩增和肝细胞成熟。如图4所示,iPS细胞分化第3天,细胞分化到内胚层阶段,免疫荧光可检测到内胚层特异maker SOX17蛋白;分化第13天,细胞分化到肝前体阶段,免疫荧光可检测到肝祖细胞特异maker AFP蛋白;分化第21天,得到肝样细胞,免疫荧光可检测到肝细胞特异maker ALB蛋白。
实施例43D肝类器官培养
将实施例3获得的2D肝样细胞培养成3D肝类器官,3D肝类器官培养步骤如下:
1)将分化好的肝样细胞用Accutase消化15min,加入500μL DMEM/F12培养基重悬,300g离心3min,弃上清;
2)用未稀释的Matrigel重悬细胞,接种到24孔板中,每孔约50μL,置于37℃细胞培养箱20min,待Matrigel凝固,加入500μL已配制好的肝类器官培养基,每3天换液;
3)14天后,按1:4比例进行传代,每3天换液。
肝类器官培养基组成:
AdDMEM/F12(Thermo Scientific),0.5%青霉素-链霉素(Penicillin-Streptomycin),1%GlutaMAX,10mM HEPES,1%B27 minus vitamin A,15%R-spodin1条件培养基(R-spodin1-conditioned medium),3-sChIR99021(Sigma),10mM烟酰胺(nicotinamide)(Sigma),10nM胃泌素(gastrin)(Sigma),50ng mL-1EGF(Peprotech),20ngmL-1TGF-α(Peprotech),100ng mL-1FGF7(Peprotech),100ng mL-1FGF-10(Peprotech),50ngmL-1HGF(Peprotech),2mM A83-01(Tocris),10μM Y-27632、1μM地塞米松(dexamethasone)(Sigma),10ng mL-1OncostatinM。
图5显示了由上述方法获得的患者3D肝类器官肝细胞makerALB蛋白、AFP蛋白的鉴定结果。
实施例5肝细胞铁过载模型以及肝类器官铁过载模型的构建
采用实施例3和4中同样的方法,区别仅在于iPS细胞为实施例2中经CRISPR/cas9基因修复技术修复后的iPS细胞,从而获得修复2D肝样细胞和修复3D肝类器官。采用实施例3和4中同样的方法,区别仅在于iPS细胞源自正常人的成纤维细胞(iPS细胞获取方法同实施例1),从而获得正常人2D肝样细胞和正常人3D肝类器官。利用实施例3中获得的病人肝样细胞和实施例4中获得的病人肝类器官,以及病人修复肝样细胞和病人修复肝类器官、正常人肝样细胞和正常人肝类器官,构建FAC诱导的肝细胞、肝类器官铁过载疾病模型。
FAC是柠檬酸铁铵,是一种常用的诱导细胞铁过载的化合物。图6A显示不同浓度FAC处理下,尤其是FAC为大于1mM浓度下,病人肝类器官比正常人和病人修复的类器官中细胞死亡更多,病人肝细胞对铁过载导致的细胞死亡更敏感。铁死亡是铁导致的细胞脂质ROS增加引起的细胞死亡方式,通过大量实验证实,病人肝细胞铁过载的死亡方式为铁死亡。图6B显示了用不同FAC浓度处理下,正常人、病人、病人修复肝样细胞细胞存活的统计结果,表明5mM FAC浓度下,病人肝样细胞铁过载模型相比正常人、病人修复肝样细胞铁过载模型细胞存活率更低。
1、用柠檬酸铁铵(FAC)诱导肝类器官铁过载步骤:
1)用指定浓度0、1、5、10mM的FAC处理正常人、病人和病人修复的肝类器官器官72小时,观察到病人肝类器官与正常对照组和修复组相比,在5mM FAC浓度下细胞显著死亡。
2)细胞死亡检测方法:使用SYTOX Green(Invitrogen)检测细胞死亡。用浓度0、1、5、10mM的FAC处理类器官后,PBS洗三次,用30nMSYTOX Green染色,置于细胞培养箱进行孵育20min,PBS洗三次,用荧光显微镜观察,使用image J软件定量死亡细胞(SYTOX Green阳性)与总细胞的比例。
2、用柠檬酸铁铵诱导肝样细胞铁过载步骤:
1)肝样细胞铁过载模型构建:用指定浓度0、2.5、5mM的FAC处理正常人、病人和病人修复的肝样细胞48小时,观察到病人肝样细胞与正常对照组和修复组相比,在5mM FAC浓度下细胞显著死亡。
2)细胞死亡检测方法:使用CCK8(Beyotime)检测细胞死亡。将肝样细胞消化并计数,按1×106数量接种在96孔板中。第二天待细胞完全贴壁后,用指定浓度0、2.5、5mM的FAC处理48小时。将10μL CCK8试剂添加到每个孔中,置于细胞培养箱中孵育4小时,然后通过酶标仪在450nm下进行检测。
实施例6肝样细胞谷胱甘肽(GSH)含量、游离铁含量、铁蛋白和溶酶体共定位检测
1、肝样细胞谷胱甘肽(GSH)含量检测:使用谷胱甘肽比色测定试剂盒(Bivison)测量细胞内GSH水平。收集1×106个肝样细胞,然后平均分为两部分,按照试剂盒说明书,一部分用于测定还原型GSH,另一种用检测总GSH。
采用上述方法分别测定正常人肝样细胞、病人肝样细胞以及病人修复肝样细胞中还原型GSH/总GSH的值。图7A结果说明病人肝样细胞还原型GSH含量低于正常人和病人修复肝样细胞。
2、游离铁含量检测:使用荧光探针Phen Green SK(Invitrogen)对检测细胞内游离铁含量。将FAC处理(5mM)和未处理的肝样细胞用5μM的Phen Green SK在细胞培养箱中孵育15min,吸掉培养基,PBS洗三次,将肝样细胞消化并收集起来,使用滤网过滤成单细胞悬液,用流式细胞仪进行分析。
图7B中结果显示病人肝样细胞游离铁含量高于正常人和病人修复肝样细胞。
3、铁蛋白和溶酶体共定位检测:使用免疫荧光法检测铁蛋白和溶酶体共定位情况。FAC(5mM)处理48h后,使用0.1μMLysoTracker对溶酶体进行标记,置于细胞培养箱1小时。后续PBS洗三次,进行常规的免疫荧光实验。
图7C中结果显示相对于正常人和病人修复肝样细胞,病人肝样细胞中铁蛋白和溶酶体共定位更高。
通过大量实验证实,病人肝细胞的铁蛋白进入溶酶体降解,导致铁释放到胞质,引发病人肝细胞铁死亡。
实施例7药物筛选
药物筛选具体步骤如下:
1、细胞脂质ROS检测:5mM FAC、5mM FAC+0.5mM DFO、5mM FAC+10μM Fer-1、5mMFAC+5mM NAC分别处理正常人、病人和病人修复的肝样细胞24小时后,使用C11-BODIPY(581/591)(Invitrogen)进行细胞脂质ROS检测。2μM C11-BODIPY(581/591)染色细胞30min,使用流式细胞仪进行检测。
图8A显示了分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝样细胞的脂质ROS检测的结果,图8B显示了分别用FAC+DMSO、FAC+NAC处理的正常人、病人和病人修复的肝样细胞的脂质ROS检测的结果,其中DMSO组为对照。结果显示DFO、Fer-1、NAC可以降低细胞脂质ROS,从而拯救病人肝样细胞和肝类器官的铁死亡。
2、细胞存活率检测方法:使用CCK8(Beyotime)检测细胞死亡。将肝样细胞消化并计数,按1×106数量接种在96孔板中。第二天待细胞完全贴壁后,用5mM FAC、5mM FAC+0.5mM DFO、5mM FAC+10μM Fer-1、5mM FAC+5mM NAC分别处理正常人、病人和病人修复的肝样细胞48小时后。将10μL CCK8试剂添加到每个孔中,置于细胞培养箱中孵育4小时,然后通过酶标仪在450nm下进行检测,统计细胞存活率。
图8C显示了分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝样细胞存活率的结果,图8D显示了分别用FAC+DMSO、FAC+NAC处理的正常人、病人和病人修复的肝样细胞存活率的结果,其中DMSO组为对照。结果显示DFO、Fer-1、NAC可以提高肝样细胞存活率。
3、使用SYTOX Green(Invitrogen)检测细胞死亡:用5mM FAC、5mM FAC+0.5mMDFO、5mM FAC+10μM Fer-1、5mM FAC+5mM NAC分别处理正常人、病人和病人修复的肝类器官72小时后,PBS洗三次,用30nM SYTOX Green染色,置于细胞培养箱进行孵育20min,PBS洗三次,用荧光显微镜观察,使用image J软件定量死亡细胞(SYTOX Green阳性)与总细胞的比例。
图8E显示了分别用FAC+DMSO、FAC+DFO和FAC+Fer-1处理的正常人、病人和病人修复的肝类器官细胞死亡的结果,其中DMSO组为对照,图8F显示了分别用FAC、FAC+NAC处理的正常人、病人和病人修复的肝类器官细胞死亡的结果。结果显示DFO、Fer-1、NAC能够降低肝类器官细胞死亡。
发明人发现DFO通过螯合细胞内的铁,从而抑制细胞铁死亡。Fer-1是抗氧化剂,由于铁死亡是铁引起细胞脂质ROS增加导致的死亡,所以抗氧化剂Fer-1可以抑制铁死亡。NAC是GSH的前体,补充NAC可以抵抗脂质ROS,从而抑制铁死亡。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
<110> 中国科学院广州生物医药与健康研究院
<120> 一种肝脏类器官模型及其建立方法、用途以及治疗肝细胞铁死亡的药物组合物
<130> BI3210222
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 病人1的sgRNA1对应的DNA序列
<400> 1
caccgtctga tgaacattcc agtgc 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 病人1的sgRNA2对应的DNA序列
<400> 2
aaacgcactg gaatgttcat cagac 25
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 病人2的sgRNA1对应的DNA序列
<400> 3
caccgccttc gatggagagc cttcg 25
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 病人2的sgRNA2对应的DNA序列
<400> 4
aaaccgaagg ctctccatcg aaggc 25
Claims (8)
1.一种2D肝样细胞铁过载模型的建立方法,其特征在于,所述建立方法包括:
(1)利用离体iPS细胞分化获得2D肝样细胞;
(2)利用柠檬酸铁铵与所述2D肝样细胞接触,以便获得2D肝样细胞铁过载模型,
其中,所述iPS细胞源自哺乳动物的皮肤成纤维细胞;
所述iPS细胞中含有与线粒体DNA缺失综合征相关的DGUOK基因突变;
所述柠檬酸铁铵的浓度为5 mM。
2.根据权利要求1所述的建立方法,其特征在于,所述iPS细胞通过以下方法获取:
1)将含有OCT4、SOX2、KLF4、c-MYC基因的病毒感染哺乳动物的皮肤成纤维细胞;
2)对感染后第六天的细胞进行分盘:将细胞用0.25%胰酶消化传代,按照每盘30-40×104个细胞/6 cm盘的密度接种细胞,用4mL 再编程培养基2进行培养,每天更换培养基,感染后16天将所述再编程培养基2更换为mTeSR培养基继续培养,其中,接种细胞所用的盘为用Matrigel包被的盘;
3)根据培养获得的克隆的形态挑取iPS细胞克隆;
其中,所述再编程培养基2包括500mL F12培养基、10ml ITS、32mg维生素c、2.5g NaCl、5μg bFGF以及100μM丁酸钠。
3.一种利用权利要求1或2所述的建立方法获得的2D肝样细胞铁过载模型。
4.一种3D肝脏类器官铁过载模型的建立方法,其特征在于,所述建立方法包括:
1)对权利要求1或2所述的建立方法获得的2D肝样细胞进行3D肝脏类器官培养,以便获得3D肝脏类器官;
2)利用柠檬酸铁铵与所述3D肝脏类器官接触,以便获得3D肝脏类器官铁过载模型,
其中,所述柠檬酸铁铵的浓度为5 mM;
进行所述3D肝脏类器官培养的方法包括:
(Ⅰ)将所述2D肝样细胞用Accutase消化10-15 min,加入DMEM/F12培养基重悬,弃上清;
(Ⅱ)用Matrigel重悬细胞,接种到培养板中,置于细胞培养箱培养20-30 min,待Matrigel凝固,加入肝类器官培养基,每3天换液一次;
(Ⅲ)在培养箱中培养14天后,按1:4比例进行传代,每3天换液一次,以便获得3D肝脏类器官。
5.根据权利要求4所述的建立方法,其特征在于,所述肝类器官培养基组成:
AdDMEM/F12、0.5%青霉素-链霉素、1%GlutaMAX、10 mM HEPES、1% B27 minusvitamin A、15%R-spodin1条件培养基、3μM ChIR99021、10 mM烟酰胺、10 nM胃泌素、50 ngmL-1 EGF、20 ng mL-1TGF-α、100 ng mL-1 FGF7、100 ng mL-1 FGF10、50 ng mL-1 HGF、2 mMA83-01、10 µM Y-27632、1 µM地塞米松,10 ng mL-1 Oncostatin M。
6.一种利用权利要求4或5所述的建立方法获得的3D肝脏类器官铁过载模型。
7.权利要求1或2所述的建立方法获得的2D肝样细胞铁过载模型、权利要求3所述的2D肝样细胞铁过载模型、权利要求4或5所述的建立方法获得的3D肝脏类器官铁过载模型、权利要求6所述的3D肝脏类器官铁过载模型在筛选药物中的用途,其特征在于,所述药物用于治疗肝细胞铁过载或铁死亡。
8.一种筛选治疗肝细胞铁过载或铁死亡的药物的方法,其特征在于,所述方法包括:
将权利要求1或2所述的建立方法获得的2D肝样细胞铁过载模型、权利要求3所述的2D肝样细胞铁过载模型、权利要求4或5所述的建立方法获得的3D肝脏类器官铁过载模型、权利要求6所述的3D肝脏类器官铁过载模型中的至少之一与待测药物接触,所述2D肝样细胞和/或3D肝脏类器官中细胞内的游离铁含量降低、细胞还原型GSH含量升高、细胞中铁蛋白和溶酶体共定位降低、细胞脂质ROS降低中的至少之一,是所述待测药物能够治疗肝细胞铁过载或铁死亡的指示。
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