CN113717928B - 基于框架核酸材料构建3d肝芽类器官的方法和应用 - Google Patents
基于框架核酸材料构建3d肝芽类器官的方法和应用 Download PDFInfo
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Abstract
本发明公开了基于框架核酸材料构建3D肝芽类器官的方法和应用，属于生物医药领域。该构建方法包括：基于框架核酸材料(FNAs)设计细胞连接器(FNA‑Linker)，并分别与肝实质细胞和肝内非实质细胞混合进行孵育使FNA‑Linker固定于细胞膜表面，孵育后的两种细胞按比例混合均匀后倒置悬滴培养，在悬滴和FNA‑Linker的DNA互补配对连接得到3D肝芽类器官，其内包含肝实质细胞和肝内非实质细胞，通过其构建慢性肝病体外模型如非酒精性脂肪肝病体外模型和肝纤维化体外模型，可以替代慢性肝病动物模型用于筛选治疗慢性肝病药物。
Description
技术领域
本发明属于生物医药技术领域，具体涉及基于框架核酸材料构建3D肝芽类器官的方法和应用。
背景技术
肝病一直以来都是人类健康的重要杀手，全球每年有超过200万人死于肝病，其中肝硬化并发症和病毒性肝炎肝细胞癌是主要致死因素，慢性肝病由于病期长、发病率高已成为全球重要的经济负担。随着生活水平的提高、饮食方式的改变，酒精性肝病(ALD)和非酒精性脂肪肝病(NAFLD)的发病率逐年上升，病原因素会刺激肝内纤维组织增生，长期的纤维组织增长、胶原蛋白的积累会导致肝小叶结构被破坏形成假小叶等，如进一步发展会恶化为不可逆转的肝硬化甚至肝癌。慢性肝病的病理发展通常是一个漫长的过程，并且在发展为肝硬化之前都是可逆转的，这一时期理论上是治疗慢性肝病的最佳时期，抑制肝纤维化的发展可以有效地降低肝硬化和肝癌的发生率，降低慢性肝病的致死率。但是目前仍未研发出能够有效抑制肝纤维化的临床药物，至今仍有超过50％–80％的慢性肝病患者转化为肝硬化和原发性肝癌，因此揭示慢性肝病的发展机制、研发高效的抗纤维化药物是应对慢性肝病的当务之急。
慢性肝病疾病动物模型是研究疾病致病机制和筛选治疗药物的必要工具，目前实验动物模型在慢性肝病机制研究和临床药物研发的应用中存在以下几个弊端：①研究周期长；②不利于连续监测；③不利于高通量筛选；④种属差异；⑤经济成本高，这些因素严重限制慢性肝病致病机制的研究和治疗药物的筛选。3D肝芽类器官可以在细胞类型、空间结构等方面更精确的模拟人类肝脏，为研究慢性肝病的发生发展、构建生物样本库、筛选药物等应用提供理想的研究工具。3D肝芽类器官具有适合大规模培养、可以连续监测、没有种属差异、继承患者个体遗传信息等优势，将其应用在机制研究和药物筛选中，可以更精准的模拟病理过程、缩短研究研发周期。目前体外3D肝芽类器官的构建技术主要包括干细胞诱导分化、多细胞联合培养、生物材料模具和3D打印等，但是均受制于干细胞分化技术、生物材料模具等因素，细胞组成单一，只有肝实质细胞，缺乏慢性肝病的关键效应细胞——肝星状细胞，无法作为慢性肝病的研究模型。Takebe等人建立的3D肝芽(Liver Bud)，包含血管内皮和间充质细胞，细胞类型有所扩展，但是该方法需要复杂且昂贵的3D基质胶膜具，为了粘合细胞团需要引入大量内源肝脏环境中不存在的间充质干细胞，并且需要7天左右的时间才能形成稳定的3D肝芽结构，其技术复杂、成本高也不利于大规模应用。复杂模具如瑞士Insphero公司开发的特殊双层结构悬滴培养板或Corning公司等开发的超低吸附球形底培养板用于3D肝芽的构建，但其成本同样高昂、构建周期仍然较长。因此，需要开发一种构建高效、低成本、可灵活控制细胞组分的3D肝芽类器官的新方法。
发明内容
本发明的主要目的是提供基于框架核酸材料构建3D肝芽类器官的方法，采用基于框架核酸材料(FNAs)设计的细胞连接器(FNA-Linker)将肝实质细胞和肝内非实质细胞按比例在体外培养液悬滴中快速组装形成3D肝芽类器官，无需基质胶和复杂模具，周期和成本大幅降低，适合大规模培养。
本发明的另一目的是提供通过上述构建方法得到的3D肝芽类器官，其包含肝实质细胞和肝内非实质细胞，作为慢性肝病体外模型可以连续监测且没有种属差异，克服现有的慢性肝病动物模型存在不利于连续监测和高通量筛选、研究周期长、种属差异大和经济成本高的问题。
本发明的再一目的是提供上述3D肝芽类器官在制备慢性肝病体外模型或筛选治疗慢性肝病药物中的应用。
为实现上述目的，本发明采用如下技术方案：
本发明提供基于框架核酸材料构建3D肝芽类器官的方法，采用基于框架核酸材料设计的细胞连接器(FNA-Linker)将肝实质细胞和肝内非实质细胞按比例在体外培养液悬滴中快速组装形成3D肝芽类器官，包括以下步骤：
(1)FNA-Linker分别与肝实质细胞和肝内非实质细胞混合后进行孵育，使FNA-Linker固定于细胞膜表面；
(2)步骤(1)中细胞膜表面固定有FNA-Linker的肝实质细胞和肝内非实质细胞按比例混合均匀，移液至培养板盖上制作20–35μl的液滴，培养板孔内加入PBS溶液，将制作好的培养板盖翻扣到培养板上，得到悬滴；
(3)步骤(2)中倒置有悬滴的培养板于37℃培养12–24小时，在悬滴下表面弧度和重力的作用下，FNA-Linker中DNA互补配对连接形成3D肝芽类器官。
优选地，所述3D肝芽类器官为球体或类球体。
所述肝内非实质细胞选自肝星状细胞、血管内皮细胞、Kupffer细胞或胆管细胞中的一种或两种以上组合。
步骤(1)中，孵育时间为30min。
步骤(1)中，FNA-Linker的使用量为每万个细胞20nM。
步骤(2)中，细胞膜表面固定有FNA-Linker的肝实质细胞和肝内非实质细胞的数量比为1–20：1。
本发明还提供一种3D肝芽类器官，通过上述基于框架核酸材料构建3D肝芽类器官的方法得到。
本发明还提供所述3D肝芽类器官在制备慢性肝病体外模型或筛选治疗慢性肝病药物中的应用。
优选地，所述慢性肝病体外模型包括非酒精性脂肪肝病体外模型和肝纤维化体外模型。
优选地，所述治疗慢性肝病药物为抗肝纤维化靶点药物。
与现有技术相比，本发明具有以下有益效果：
(1)现有技术中3D类肝器官通常需要一周左右分化发育时间，本发明中基于框架核酸材料构建3D肝芽类器官只需12–24小时即可将不同类型的细胞组装形成3D类肝器官，且无需基质胶和复杂模具如干细胞培养试剂、基质胶和特殊3D培养板，周期和成本大幅降低，适合大规模培养。
(2)现有技术中iPSC或原代肝细胞诱导分化培养所获的类器官无法包含其他胚层的效应细胞，本发明可以采用来自不同胚层的细胞，细胞类型多，且可根据应用目的将不同类型的细胞按比例组装在一起，不受干细胞分化限制，灵活度高。
(3)本发明中3D肝芽类器官包含肝实质细胞和肝内非实质细胞，作为慢性肝病体外模型可以连续监测且没有种属差异，克服了现有的慢性肝病动物模型存在不利于连续监测和高通量筛选、研究周期长、种属差异大和经济成本高等问题。
(4)与癌症患者不同，慢性肝病患者通常不会进行大量病灶样本采集进而分离其中干细胞去构建类器官，通过干细胞分化获得类器官也无法反映患者个体肝脏的环境影响因素，本发明可将少量肝穿刺活检样本在体外重构为多个类肝器官，即通过微量临床活检样本得到慢性肝病患者的生物银行，真实反映患者肝内细胞病变及结构组成信息，为精准医疗提供工具。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更显著：
图1是本发明中构建3D肝芽类器官的技术原理示意图。
图2是本发明中构建3D肝芽类器官的技术路线示意图。
图3是本发明中基于FNA-Linker形成3D肝芽类器官的过程变化。
图4是3D肝芽类器官的鉴定图片，箭头所示为3D肝芽类器官。
图5是3D肝芽类器官转移至6孔培养板的鉴定图片，细胞团不会散开，箭头所示为3D肝芽类器官。
图6是实施例1中3D非酒精性脂肪肝病体外模型中甘油三酯(a)、酶AST和ALT(b)分泌情况的检测结果。
图7是实施例1中3D非酒精性脂肪肝病体外模型通过qPCR检测脂质形成、运输以及炎症相关的基因表达。
图8是实施例2中非酒精性脂肪肝病体外模型用于验证化合物药效的检测结果。
图9是实施例3中3D肝芽类器官用于构建肝纤维化体外模型的检测结果。
图10是实施例4中3D肝芽类器官用于抗肝纤维化靶点药物高通量筛选的检测结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。
以下实施例中肝实质细胞和肝内非实质细胞来源于人，如肝病肝穿刺活检样本。
如图1所示，构建3D肝芽类器官的技术原理为：肝实质细胞和肝内非实质细胞分别与基于框架核酸材料设计的细胞连接器FNA-Linker A和FNA-Linker B进行孵育，FNA-Linker A和FNA-Linker B分别固定到肝实质细胞和肝内非实质细胞的细胞膜表面，FNA-Linker A和FNA-Linker B上的DNA序列互补配对将不同细胞粘合到一起形成细胞团块，然后通过倒置悬滴培养法对混合细胞进行培养，在悬滴下表面弧度和重力的作用下细胞团块形成球体或类球体结构的3D肝芽类器官。
如图2所示，构建3D肝芽类器官的步骤为：(1)将基于框架核酸材料设计的细胞连接器FNA-Linker A与肝实质细胞混合，室温孵育30分钟，使FNA-Linker A固定于细胞膜表面，同时将基于框架核酸材料设计的细胞连接器FNA-Linker B与肝内非实质细胞的混合液混合，室温孵育30分钟，使FNA-Linker B固定于细胞膜表面；(2)孵育完成后，将携带有FNA-Linker A的肝实质细胞和FNA-Linker B的肝内非实质细胞混合均匀，用移液器在培养板盖上制作20–30μl的液滴，培养板孔内加入PBS溶液，将制作好的培养板盖小心翻转扣到培养板上制作悬滴，倒置有悬滴的培养板放入37℃培养箱培养12–24小时，在悬滴下表面弧度和重力的作用下FNA-Linker中的DNA互补配对连接形成3D肝芽类器官结构(图3)。
3D肝芽类器官的鉴定：悬滴培养12–24小时后检查3D肝芽类器官的形成情况，取出悬滴培养板，发现每个悬滴中都出现肉眼可见的白色球形结构(图4)。顺时针晃动培养板，未加入FNA-Linker的悬滴内细胞(对照组)迅速散开，加入FNA-Linker的悬滴内细胞(实验组)团稳定存在，用移液器轻轻吹打也不会散开(图5)，得到的3D肝芽类器官可移入球型底96孔培养板继续培养或者直接用于构建慢性肝病体外模型。
下面通过具体实验例来进一步说明本发明的技术效果。
实验例1 3D肝芽类器官用于构建非酒精性脂肪肝病体外模型
采用基于框架核酸材料构建3D肝芽类器官后，调节细胞空间排布并通过体外脂肪酸诱导构建非酒精性脂肪肝病体外模型，步骤如下：
用含10％FBS的完全型培养基重悬所有细胞，将1万个绿色荧光蛋白GFP标记的肝实质细胞与20nM的FNA-Linker A混合，室温孵育30分钟；将5000个红色荧光蛋白mCherry标记的肝星状细胞与10nM的FNA-Linker B混合，室温孵育30分钟；将携带两种FNA-Linker的细胞混合均匀，按照每团100μl的培养体系将细胞混合孵育，置于37℃、5％CO2恒温培养箱内培养，12小时即可形成球形3D肝芽。3D肝芽类器官形成后，将25mM的棕榈酸、25mM的油酸与25％的牛血清蛋白溶液混合，在37℃水浴锅内恒温过夜，使得两种脂肪酸结合到牛血清蛋白上。随后用含10％FBS的完全型培养基将25mM的棕榈酸油酸储液稀释50倍，制成0.5mM的棕榈酸油酸脂性培养基。用0.5mM的棕榈酸油酸培养基在37℃、5％CO2恒温培养箱内培养3D肝芽类器官24小时、96小时、168小时，在不同培养时间点收集3D肝芽类器官与培养上清进行检测。三个时间点分别收取3D肝芽类器官，用PBS润洗肝芽类器官去除脂性培养基对检测结果的干扰，随后裂解肝芽类器官内细胞并抽提胞内甘油三酯进行检测，结果发现3D肝芽类器官内甘油三酯含量呈逐步增高趋势(图6a)；分别在三个时间点收取培养上清，检测损伤相关的酶AST和ALT分泌情况，结果发现AST及ALT水平的升高，标志着脂肪变化所造成的肝细胞损伤(图6b)；用qPCR检测一系列与脂质形成、运输以及炎症相关的基因表达情况，结果证明与脂质形成、炎症因子相关的基因转录水平在造模96小时后均有显著提高(图7)，证明体外含脂肪酸培养环境可诱导混合孵育的3D肝芽类器官形成非酒精性脂肪性肝病病理改变。
实施例2非酒精性脂肪肝病体外模型用于验证化合物药效
将1万个绿色荧光蛋白GFP标记的肝实质细胞与5000个红色荧光蛋白mCherry标记的肝星状细胞按上述方式混合，用0.5mM的棕榈酸油酸培养基培养混合孵育3D肝芽类器官96小时，得到非酒精性脂肪性肝病模型(即3D非酒精性脂肪性肝病类球体)。造模成功后撤去脂性培养基，造模组与对照组均采用含10％FBS的完全型培养基进行后续培养。同时选取对于NASH脂质形成有抑制作用的小分子抑制剂Elafibranor，按50μM的终浓度对3D非酒精性脂肪性肝病类球体进行48小时加药处理，50μM Elafibranor作用于上述肝病类球体48小时后，收集并润洗肝芽，发现类球体内的甘油三酯水平明显下降(图8a)。通过qPCR验证一系列与脂质形成、运载以及炎症相关的基因表达水平，结果同样证明加入Elafibranor后，肝芽内相关基因水平呈显著下降趋势(图8b，图8c)，证明非酒精性脂肪肝病体外模型可用于NASH治疗药物的药效验证。
实施例3 3D肝芽类器官用于构建肝纤维化体外模型
采用基于框架核酸材料构建3D肝芽类器官后，通过体外激活纤维化进程构建肝纤维化体外模型，步骤如下：
将1万个绿色荧光蛋白GFP标记的L02细胞、2000个红色荧光蛋白mCherry标记的LX2和5000个不带荧光标记的HUVEC按上述方式制作肝纤维化类肝器官体外模型。分别在培养24小时和168小时收集类器官，收集的肝芽进行冰冻切片用于后续的染色，通过天狼猩红染色标记胶原，证实随着培养时间的延长，胶原的沉积逐渐增多(图9a)；用免疫荧光标记星状细胞激活标志物αSMA，发现αSMA水平随时间增多(图9b)；对团块内纤维化相关的基因进行qPCR检测，证明纤维化相关基因随时间延长而显著增高(图9c)，说明激活纤维化后的不同时间点可获得程度不同的纤维化3D肝芽类肝器官。
实施例4 3D肝芽类器官用于抗肝纤维化靶点药物的高通量筛选
按照实施例3的方法制作肝纤维化3D肝芽类器官体外模型后，将小分子数据库筛选出的与PDK1靶点结构相似的潜在小分子药物按照1μM、10μM的浓度对肝纤维化类肝器官加药处理72小时(图10a)，用高内涵成像技术统计红色荧光mCherry与绿色荧光GFP的荧光值，根据高内涵成像统计出的绿色荧光值数据，先按照均值减去一个标准差为界限，去除绿色荧光值低于此界限的所有的药物，选择对肝实质细胞无毒性损伤的小分子(图10b)。按照红色荧光值数据，将红色荧光值低于对照组的所有药物，根据红色荧光值从低到高进行排序，筛选出能抑制星状细胞增殖的药物，排在前四位的分别是OSU03012(37-10)、阿西替尼(4-10)、BX517(35-10)及维生素C(3-1)(图10c)。qPCR检测几种药物作用后肝芽内纤维化相关基因的表达情况，结果表明上述四种药物均不同程度地降低了纤维化相关基因的表达，其中阿西替尼效果显著，可为临床药物治疗提供新的思路(图10d)，证明通过批量制作肝纤维化3D肝芽类器官体外模型可以高通量筛选特定位点的潜在药物。
Claims (10)
1.基于框架核酸材料(FNAs)构建3D肝芽类器官的方法，其特征在于，采用基于框架核酸材料设计的FNA-Linker将肝实质细胞和肝内非实质细胞按比例在体外培养液悬滴中快速组装形成3D肝芽类器官；所述FNA-Linker包括FNA-Linker A和FNA-Linker B，所述FNA-Linker A和FNA-linker B中的Linker存在互补DNA，可令FNA-Linker A和FNA-Linker B相互连接；具体包括以下步骤：
(1)FNA-Linker A和FNA-Linker B分别与肝实质细胞和肝内非实质细胞混合后进行孵育，使FNA-Linker A和FNA-Linker B固定于细胞膜表面；
(2)步骤(1)中细胞膜表面固定有FNA-Linker的肝实质细胞和肝内非实质细胞按比例混合均匀，移液至培养板盖上制作20-35μl的液滴，培养板孔内加入PBS溶液，将制作好的培养板盖翻扣到培养板上，得到悬滴；
(3)步骤(2)中倒置有悬滴的培养板于37℃培养12-24小时，在悬滴下表面弧度和重力的作用下，FNA-Linker中DNA互补配对连接形成3D肝芽类器官。
2.根据权利要求1所述基于框架核酸材料构建3D肝芽类器官的方法，其特征在于，所述3D肝芽类器官为球体或类球体。
3.根据权利要求1所述基于框架核酸材料构建3D肝芽类器官的方法，其特征在于，所述肝内非实质细胞选自肝星状细胞、血管内皮细胞、Kupffer细胞或胆管细胞中的一种或两种以上组合。
4.根据权利要求1所述基于框架核酸材料构建3D肝芽类器官的方法，其特征在于，步骤(1)中，孵育时间为30min。
5.根据权利要求1所述基于框架核酸材料构建3D肝芽类器官的方法，其特征在于，步骤(1)中，FNA-Linker的使用量为每万个细胞20nM。
6.根据权利要求1所述基于框架核酸材料构建3D肝芽类器官的方法，其特征在于，步骤(2)中，细胞膜表面固定有FNA-Linker的肝实质细胞和肝内非实质细胞的数量比为1–20：1。
7.一种3D肝芽类器官，其特征在于，通过权利要求1至6任一项所述基于框架核酸材料构建3D肝芽类器官的方法得到。
8.权利要求7所述的3D肝芽类器官在制备慢性肝病体外模型或筛选治疗慢性肝病药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述慢性肝病体外模型包括非酒精性脂肪肝病体外模型和肝纤维化体外模型。
10.根据权利要求8所述的应用，其特征在于，所述治疗慢性肝病药物为抗肝纤维化靶点药。
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