肝脏类器官在肝脏疾病模型与移植治疗中的应用潜力

doi:10.12307/2025.359

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (8): 1684-1692.doi: 10.12307/2025.359

• 组织构建综述 tissue construction review • 上一篇下一篇

肝脏类器官在肝脏疾病模型与移植治疗中的应用潜力

袁维勃 1，2，3，刘婵1，2，3，4，5，余丽梅1，2，3，4，5

遵义医科大学附属医院，1贵州省细胞工程重点实验室，3贵州省干细胞临床应用与新药研发工程研究中心，4贵州省生物治疗人才基地，5遵义市干细胞与再生医学工程研究中心，贵州省遵义市 563003；2遵义医科大学教育部省部共建组织损伤修复与再生医学协同创新中心，贵州省遵义市563000

收稿日期:2024-03-13 接受日期:2024-05-17 出版日期:2025-03-18 发布日期:2024-07-06
通讯作者: 余丽梅，博士，教授，博士生导师，遵义医科大学附属医院，贵州省细胞工程重点实验室，贵州省干细胞临床应用与新药研发工程研究中心，贵州省生物治疗人才基地，遵义市干细胞与再生医学工程研究中心，贵州省遵义市563003；遵义医科大学教育部省部共建组织损伤修复与再生医学协同创新中心，贵州省遵义市 563000
作者简介:袁维勃，男，1999年生，陕西省咸阳市人，汉族，遵义医科大学在读硕士，从事干细胞的肝病治疗研究。
基金资助:
2023年国家自然科学基金地区科学基金项目(82360047)，项目负责人：余丽梅

Potential application of liver organoids in liver disease models and transplantation therapy

Yuan Weibo1, 2, 3, Liu Chan1, 2, 3, 4, 5, Yu Limei1, 2, 3, 4, 5

1Guizhou Key Laboratory of Cell Engineering, 3Guizhou Stem Cell Clinical Application and New Drug Research and Development Engineering Research Center, 4Guizhou Biotherapy Talent Base, 5Zunyi Stem Cell and Regenerative Medicine Engineering Research Center, Affiliated Hospital of Zunyi Medical University, Zunyi 563003, Guizhou Province, China; 2Collaborative Innovation Center for Tissue Injury Repair and Regenerative Medicine Co-Built by Province and Ministry of Education, Zunyi Medical University, Zunyi 563000, Guizhou Province, China

Received:2024-03-13 Accepted:2024-05-17 Online:2025-03-18 Published:2024-07-06
Contact: Yu Limei, MD, Professor, Doctoral supervisor, 1Guizhou Key Laboratory of Cell Engineering, 3Guizhou Stem Cell Clinical Application and New Drug Research and Development Engineering Research Center, 4Guizhou Biotherapy Talent Base, 5Zunyi Stem Cell and Regenerative Medicine Engineering Research Center, Affiliated Hospital of Zunyi Medical University, Zunyi 563003, Guizhou Province, China; 2Collaborative Innovation Center for Tissue Injury Repair and Regenerative Medicine Co-Built by Province and Ministry of Education, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
About author:Yuan Weibo, Master candidate, 1Guizhou Key Laboratory of Cell Engineering, 3Guizhou Stem Cell Clinical Application and New Drug Research and Development Engineering Research Center, Affiliated Hospital of Zunyi Medical University, Zunyi 563003, Guizhou Province, China; 2Collaborative Innovation Center for Tissue Injury Repair and Regenerative Medicine Co-Built by Province and Ministry of Education, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
Supported by:
2023 Regional Science Foundation Project of National Natural Science Foundation of China, No. 82360047 (to YLM)

摘要/Abstract

摘要：

文题释义：
肝脏类器官：是由具有干性的细胞作为种子细胞，辅助以细胞外基质和所需的细胞因子，遵循肝脏发育过程从而进行自组装并且在空间上重新排列成三维有序结构。
肝脏疾病：是发生在肝脏的受遗传因素或者环境因素影响的疾病总称。

背景：肝脏类器官对阐明肝脏疾病的确切病理机制及对肝脏疾病的治疗具有重要的意义。
目的：总结目前国内外该领域的基础研究，回顾以往肝脏类器官的构建、疾病建模和移植治疗的重要研究进展，探讨肝脏类器官联合组织工程技术的应用前景。
方法：检索PubMed数据库及中国知网数据库收录的相关文献，英文检索词为“liver，organoids，liver diseases”，中文检索词为“肝，类器官，肝脏疾病”，主要检索时限为2018年4月至2024年4月，通过人工阅读排除重复文献等，最终纳入94篇文献进行归纳分析。
结果与结论：①肝脏类器官构建的种子细胞主要集中于成体细胞和多能干细胞，通过辅助各种细胞因子参与信号引导作用和细胞外基质提供3D微环境，促进类器官的产生，但是整体成熟度不高，有望通过结合组织工程技术改善这一问题；②体外疾病建模方面主要在简单疾病和单基因遗传病领域研究较多，类器官高度保留患者遗传特性，有望通过结合CRISPR-Cas9基因矫正和其他新兴技术模拟更复杂的肝脏疾病并阐明更深层次的病理机制；③体内移植治疗方面，肝脏类器官能够安全有效的植入，表现出惊人的肝功能替代潜力及组织再生能力，并且还可能通过与其他组织工程材料结合达到治疗目的。
https://orcid.org/0009-0003-4868-6873(袁维勃)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 肝脏类器官, 干细胞, 种子细胞, 肝脏疾病, 疾病建模, 体内移植, 综述

Abstract: BACKGROUND: Liver organoids are of great significance to elucidate the exact pathological mechanism of liver diseases and the treatment of liver diseases.
OBJECTIVE: To summarize the basic research in this field at home and abroad, review the important research progress in the construction of liver organoids, disease modeling and transplantation therapy, and discuss the application prospect of combined tissue engineering technology of liver organoids.
METHODS: The relevant articles included in PubMed and CNKI databases were searched. The English and Chinese search terms were “liver, organoids, liver diseases.” The main search time was from April 2018 to April 2024. Duplicate literature was excluded by manual reading. Finally, 94 articles were included for review and analysis.
RESULTS AND CONCLUSION: The seed cells constructed by liver organoids are mainly concentrated in adult cells and pluripotent stem cells, which promote the generation of organoids by assisting various cytokines to participate in signal guidance and providing 3D microenvironment by extracellular matrix. However, the overall maturity is not high, which is expected to improve this problem by combining tissue engineering technology. In vitro disease modeling is mainly studied in the field of simple diseases and single-gene genetic diseases. Organoids highly retain patient genetic characteristics, and it is expected to simulate more complex liver diseases and clarify deeper pathological mechanisms by combining CRISPR-Cas9 gene correction and other emerging technologies. In vivo transplantation treatment, liver organoids can be safely and effectively implanted, showing amazing liver function replacement potential, tissue regeneration ability, and may also be combined with other tissue engineering materials to achieve therapeutic purposes.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: liver organoids, stem cell, seed cell, liver disease, disease modeling, in vivo transplantation, review

中图分类号:

袁维勃, 刘婵, 余丽梅. 肝脏类器官在肝脏疾病模型与移植治疗中的应用潜力[J]. 中国组织工程研究, 2025, 29(8): 1684-1692.

Yuan Weibo, Liu Chan, Yu Limei. Potential application of liver organoids in liver disease models and transplantation therapy[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(8): 1684-1692.

图/表（结果） 7

2.1 肝脏类器官几十年肝细胞和干细胞移植治疗肝病的探索，极大地推动了肝脏类器官研究的发展。干细胞来源的肝样细胞移植很大程度上克服了原代肝细胞难以突破的体外高质量和规模化培养及伦理限制[20]，取得了肝脏损伤修复和改善肝功能的疗效[21]，但是肝细胞毕竟不能完全替代复杂的肝脏结构和功能[22]，肝管破坏导致的胆汁分泌、转运和代谢障碍，及门脉系统的营养物质、毒物或代谢产物转运等突出问题。因此，利用肝细胞、干细胞或肝样细胞与生物材料及肝细胞再生相关因子等形成的肝脏类器官技术应运而生，成为了探讨肝脏组织发育与形成、肝脏疾病致病机制、肝脏再生、终末期肝脏疾病治疗的新策略[23]。肝脏类器官及其结合组织工程技术的重要事件的时间轴(图4)，大鼠肝细胞的三维球形聚集体可能是构建肝脏类器官的开端[24]。20世纪90年代，生物可降解支架、水凝胶和微载体等技术应用于肝脏类器官[25-27]。21世纪以来，生物反应器和三维打印技术逐渐在肝脏类器官领域得到应用[28-29]。接下来，具有血管化的肝脏类器官和基因修饰的类器官相继出现[30-31]。直到近几年，构建模式相对成熟的情况下，肝脏类器官扩展到胆道闭锁、病毒感染的类器官疾病模型[32-33]。

2.1.1 肝脏类器官的构建
(1)肝源组织成体细胞来源的肝脏类器官：虽然不同的种子细胞在肝脏类器官的构建中遵循不同的构建路径，但是它一定是在拥有种子细胞、细胞外基质和相应的信号通路分子3个地基的基础之上，细胞通过模拟肝脏发育过程从而进行自组装并且在空间上重新排列成有序结构[34]。2001年，MICHALOPOULOS等[35]从大鼠肝脏中分离肝细胞，利用胶原包被，辅助肝细胞生长因子和表皮生长因子等简单的细胞因子形成了具有肝结构特征的组织。近年来，利用原代肝细胞作为种子细胞与多种细胞因子和生物材料联合，构建肝类器官也取得了很大的进展[36-37]。研究人员将分离的原代肝细胞悬浮在Matrigel中，辅助以Wnt激动剂(R-spondin1和CHIR99021)、表皮生长因子、成纤维生长因子7、成纤维生长因子10、肝细胞生长因子和转化生长因子β抑制剂等因子进行三维培养，接着分别用优化的肝细胞类器官培养基(人或者鼠)和胆管细胞培养基培养出肝脏类器官(图5)。肝细胞类器官表达肝细胞标志物(白蛋白、肝细胞核因子4α、细胞色素氧化

酶Cyp1a2和Cyp3a11)、胎儿肝细胞标志物甲胎蛋白、胆管细胞/祖细胞标记物角蛋白19、祖细胞标志物(Tbx3和Sox9)，在一些细胞功能基因(细胞色素P450、糖原代谢、脂质代谢、类固醇代谢、尿素循环和补体激活)出现与原代肝细胞相似的表达谱[37]。此外，肝癌类器官可以直接从手术切除或穿刺活检组织中建立，其方法遵循正常肝脏类器官的流程[38]。
(2) iPSC来源的肝脏类器官：人iPSCs是衍生类器官最常用的种子细胞来源[39]。常用的iPSCs来源肝脏类器官构建方法见图6。

一种是从iPSCs生成成熟肝细胞，随后构建肝脏类器官[40-41]。使用富含激活素 A、骨形态发生蛋白2、成纤维生长因子4、肝细胞生长因子、重组人角质细胞生长因子和抑瘤素M的培养基实现2维成熟肝细胞的分化，然后进行三维培养，加入表皮生长因子、转化生长因子α、成纤维生长因子7、成纤维生长因子10、肝细胞生长因子、转化生长因子β抑制剂A83-01、ROCK抑制剂Y-27632、地塞米松和抑瘤素M因子，最终获得肝脏类器官[40]。
另一种是先建立iPSCs分化早期的球状体，然后进一步诱导为成熟肝脏类器官[42]。使用富含激活素 A、骨形态发生蛋白4、GSK-3抑制剂 CHIR99021和成纤维生长因子4的培养基生成前肠细胞，再将前肠细胞包埋种植于含有成纤维生长因子2、表皮生长因子、血管内皮生长因子、GSK-3 抑制剂 CHIR99021和转化生长因子β抑制剂A83-01等因子的类器官形成培养基进行三维培养，类器官形成后，切换肝脏成熟培养基培养，最终形成肝脏类器官[42]。
虽然大多数方案包括多个阶段，但都是通过激活肝细胞成熟过程中的关键信号通路，进而通过特定的发育标记来验证分化效率[43]。一般来说，通过多个阶段进行更加精确的信号调控诱导细胞分化，可以得到更高的分化效率。总之，肝细胞或干细胞衍生的肝脏类器官具有强大的增殖能力，具有基因组稳定性的优势，且肝脏类器官在一定的微环境下除向肝细胞分化外，还可向胆管细胞分化[32，44]。
2.1.2 肝脏类器官的疾病模型疾病模型对于研究疾病机制和开创治疗方法是必须的，但是由于动物与人类种属和基因组的差异，动物肝脏疾病模型与人类肝脏疾病致病机制存在不同，人类疾病的组织学复杂性和遗传异质性在许多动物模型中没有得到很好的回馈[45]。目前，利用人iPSCs、胚胎干细胞、肝细胞、肝星状细胞和枯否细胞、肝内胆管细胞和肝外胆管细胞、胆囊细胞、良性胆囊腺瘤组织细胞、成人胆管衍生的双能祖细胞、原发性硬化性胆管炎患者的胆管细胞构建的肝脏类器官已应用于多种肝脏疾病的建模及致病机制研究，见表1。
2.2 肝脏类器官的体内移植肝脏类器官的终极目标是创造出可移植的肝脏组织替代受损肝脏组织，快速地恢复肝功能[60]。富马酰乙酸水解酶缺乏会导致肝功能衰竭，HUCH等[61]培养的Lgr5+细胞衍生的肝脏类器官具有成熟肝细胞的特征与功能，移植到富马酰乙酸水解酶缺乏小鼠中，肝衰竭小鼠存活率显著增加，并在肝组织中观察到主要由肝细胞核因子4α和角蛋白19阳性的成熟肝细胞组成的富马酰乙酸水解酶阳性结节。同年，TAKEBE课题组[18]将iPSCs生成内胚层细胞通过内皮细胞和间充质干细胞之间的相互作用建立的肝脏类器官移植到肠系膜上探索类器官的功能性和相容性，肝脏类器官移植48 h内与宿主血管系统建立了联系，并执行白蛋白产生和药物代谢功能，成功挽救更昔氯韦诱导的药物性急性肝衰竭，这些是科学家最早探索肝脏类器官体内移植成功治疗肝脏疾病的实验，随后肝脏类器官的体内移植应用得到更多肝脏疾病模型的验证，见表2。此外，采用含有猪肝细胞与人脐静脉内皮细胞球体的改良生物人工肝治疗猴的急性肝衰竭，明显提高了动物的生存率，降低了血氨和炎症因子水平，改善了受损肝脏的再生微环境，很好地恢复了衰竭动物的肝脏功能[62]。

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引言

肝脏疾病(非酒精性脂肪肝、肝炎、肝纤维化、肝硬化及胆管炎、胆囊炎，以及病毒、细菌感染引起的肝脏疾病和一些代谢相关遗传因素所致的肝病等)是严重影响人类健康的一大类疾病，一般要经历炎症、纤维化、肝硬化进入终末期肝病。全球每年约有200万人因重症肝病死亡[1]，许多晚期肝病唯一有效的救治方式只能是肝移植[2-3]。在中国肝脂肪变性和纤维化已经成为一大社会问
题[4-5]，并且非酒精性脂肪肝也是肝移植的第二大原因。然而，肝供体短缺、成本高、术后并发症等问题制约了肝移植的普及，肝细胞和干细胞移植成为可能替代肝脏功能和修复肝组织的治疗策略[6]。大量的研究显示，肝细胞移植在肝脏代谢紊乱及急性肝衰竭中可替代部分肝功能，干细胞则在肝脏疾病的炎症和纤维化阶段发挥治疗作
用[7-11]。
2017年以来，以成体干细胞、原代肝细胞、原代胆管细胞、胚胎干细胞或诱导多能干细胞(induced pluripotent
stem cells，iPSCs)为种子细胞建立的类器官技术，为多种组织器官的再造开启了更具潜力的再生医学新时代[12]。目前，肝类器官的研究发展迅速[13]，主要是基于成体细胞和iPSCs培养，而胚胎干细胞的研究有限[14]。已成功构建的肝实质细胞类器官[15]、胆管细胞类器官[16]、肝胆类器官和血管化肝脏类器官[17-18]，有望通过肝脏类器官技术的创新逐渐克服供体肝脏短缺的问题[19]，或给不适合肝移植的终末期肝病患者带来再生、修复严重受损肝脏组织的新希望。因而，此文主要综述了肝脏类器官在肝脏疾病模型与移植治疗方面的研究进展。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

材料方法

讨论

3 总结与展望 Summary and prospects
不同种子细胞来源的肝脏类器官虽然在功能和形态水平显示出了肝成熟潜力，但是整体成熟水平仍然不如原代肝细胞，iPSCs衍生的肝脏类器官仅能够表现出胎儿肝脏器官的相似的功能与标志分子特征，主要表现在甲胎蛋白持续表达和更成熟的细胞色素P450亚型的缺乏或活性降低[72]。研究人员不断尝试结合组织工程技术，创造更接近生理的复杂且成熟的器官系统，为了提高肝脏类器官成熟潜力，一方面通过引入人工支架或者细胞外基质构建细胞外生态位，WILLEMSE等[73]利用人肝细胞外基质水凝胶替代常规小鼠肿瘤源性基底膜提取物作为三维培养环境，虽然未提及类器官的成熟，但是胆管细胞类器官可在动态培养中扩增。也有研究人员使用纳米氧化石墨烯颗粒来修饰去细胞化肝脏支架生物物理性质的策略[74]。TAMAI等[75]使用生物反应器生成具有胶原纤维基质的三维培养环境来包裹成纤维细胞和肝祖细胞，形成的功能性肝脏类器官在移植后还能维持其获得性功能，并且观察到部分血管生成，这种方法展示了“细胞-基质”相互作用的组织工程技术对肝脏类器官的应用潜力。一方面通过添加其他细胞类型进行共培养再现肝脏微环境以及细胞间的信号传导。内皮细胞不仅促进血管生成，帮助器官发育获得氧气和营养物质，内皮细胞所分泌的特异性分化因子也参与了器官的形成[76]。将脂肪微血管内皮细胞与人多能干细胞共培养后，能够提高肝细胞的肝向分化效率，形成的肝脏类器官表现出更好的白蛋白分泌功能[65]。YOON等[77]构建的多类型细胞肝脏类器官的甲胎蛋白、细胞色素P450酶和肝窦内皮细胞功能相关基因表达增强，这可能至少预示着肝脏类器官在功能上更加成熟。另一方面，将类器官整合到器官芯片上，通过微流体系统产生物质交换和剪切应力使肝脏类器官的结构和代谢功能的复杂性得到改善[78-79]，基于此，代表生理相关性更高的集成多类器官芯片系统也将是一个重要方向[80-82]。
目前，基于从患者身上无论是利用iPSCs或者其他原代细胞构建类器官疾病模型，能够保留其遗传背景，这也是目前所有的遗传病模型都是单基因疾病的一大原因。利用肝脏类器官及建立的肝脏疾病模型，进一步探讨肝星状细胞激活与抑制、肝脏脉管系统破坏与形成、肝脏3大物质合成与代谢、肝脏疾病状态下药物、毒物生物转化等肝脏功能发挥和生理、病理机制[80-81]，不仅有助于发现肝脏疾病治疗的新靶点、新策略，还使得利用基因编辑与肝脏类器官技术结合，修复遗传缺陷所致肝脏疾病成为可能[83]。DGUOK突变患者的iPSCs构建的肝脏类器官保留了患者的遗传背景，通过CRISPR-Cas9基因编辑纠正的肝脏类器官不容易发生铁凋亡[40]。最近通过类器官内单细胞CRISPR筛选发现，Fos和Ubr5是肝细胞成熟和分化的关键因子[84]；还模拟TP53丢失和L3R249s突变等[85]，
为探讨人类肝癌发生的早期机制和干预策略，奠定了坚实基础。利用CRISPR-Cas9技术结合类器官发育，深刻认识多种调控肝脏发育与人类肝脏疾病如肝脏肿瘤发生的系列生物学特征、行为和分子事件[86]，发现细胞、药物干预肝脏疾病新靶标、新机制，建立有效治疗新方案或预测药物在人体肝脏代谢的复杂过程及肝毒性等，已开始逐渐由基础转向临床药物筛选。随着CRISPR-Cas9基因编辑技术的不断进步，未来会有更多肝脏类器官模型有望阐明基因遗传病的病理机制。另外，复杂的肝脏疾病不仅涉及遗传成分，还要考虑环境因素以及多种细胞类型都可以影响疾病表型[72]。这就要从构建肝脏类器官这个根源上结合组织工程技术进行创造性发展。
尽管干细胞和肝脏类器官的功能特征还有大量亟待充分认识，人们已逐步理解干细胞移植后需要数周时间才能产生明显的治疗效果[87]，而急性肝衰竭的治疗窗口期非常狭窄，因此，肝脏类器官的移植应用可能是快速产生治疗效应、提高急性肝衰竭治疗效果的新策略[88]。肝脏类器官移植治疗终末期肝病的动物实验中，及时发挥了肝脏组织功能，不但明显提高肝脏的糖、脂和蛋白合成、代谢功能、药物转化及解毒功能，显著提高生存率，还可减轻肝细胞受损状态，明显促进了肝脏再生与功能修复(表2)。剑桥大学等机构研究人员也首次证明了实验室培养的“迷你胆管”移植到受损人类肝脏中并修复它们的可能性[68]。此外，临床级海藻盐材料包封肝脏类器官的移植应用也被证明是安全可行的[71]，同时，这项研究将三维组织工程技术与肝脏类器官结合起来构建人造器官，并移植入体，在体内发挥正常的生理功能，这为治疗肝脏疾病提供了新的见解[69]。但是，目前大多数人源肝脏类器官移植入其他物种的做法是否得当，所得出的数据是否可用于进一步临床研究，还需要进一步考量。目前，关于肝脏类器官的临床研究有2项：一项2016年的临床试验(NCT02718235)中，研究人员以肝癌细胞建立的肝脏类器官作为一种新型的预后评估标准，目的是测试CD8和白细胞介素33阳性的效应细胞对肝癌类器官的影响，这似乎是一种基于肝脏类器官体外建模并且区别于临床的疾病风险分级工具，更好地指导临床工作。另外一项2020年的临床试验(NCT103896958)中，研究人员同样基于患者活检或手术的肿瘤组织建立肿瘤类器官，其侧重点在类器官的药物筛选技术上，实现对患者的精准医疗。总之，目前的肝脏类器官临床试验主要基于疾病模型和药物筛选，未有体内移植的相关研究。

无论是肝脏类器官的构建、体外疾病建模和体内移植治疗，都需要构建肝细胞功能更加成熟、脉管系统更为完整的肝脏类器官，进而替代肝脏移植治疗多种原因所致重症肝病。因此利用生物支架[89]、非实质细胞共培养[90]、调控生成脉管系统[91]、血管化[92]、基因调控网络分析进行精确指导[93]，构建多种器官相互影响的类器官芯片系统，形成完善的肝脏类器官制造质量标准[74]，再现机体不同器官系统之间的复杂关系，以及免疫系统的相互影响，都将十分有益于阐明肝脏疾病病理机制和确定治疗新策略，更有望提供疗效更佳的可移植的肝脏类器官[94]，见图7。随着肝脏类器官制造标准和质量的不断完善，相信肝脏类器官也将很有希望实现临床应用的转化，以肝脏类器官为基础的肝脏器官再造更是具有诱人的前景。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

肝脏类器官在肝脏疾病模型与移植治疗中的应用潜力

Potential application of liver organoids in liver disease models and transplantation therapy

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