脐带脱细胞支架培养鼠肝前体样细胞功能研究

肝脏 ›› 2020, Vol. 25 ›› Issue (8): 876-880.

脐带脱细胞支架培养鼠肝前体样细胞功能研究

景宏舒, 彭媛, 王振宇, 李伟建, 袁天杰, 张洪丹, 鄢和新, 翟博

200127 上海交通大学医学院附属仁济医院肿瘤介入科(景宏舒彭媛王振宇李伟建鄢和新翟博),麻醉科(袁天杰),上海赛立维生物科技有限公司(张洪丹)

收稿日期:2020-05-06 出版日期:2020-08-31 发布日期:2020-09-04
通讯作者: 翟博,Email:zhaiboshi@sina.com
基金资助:
国家科技重大专项课题-艾滋病和病毒性肝炎等重大传染病防治(2017ZX10203206-006-002)

Study on function of rHepLPCs cultured on decellularized human umbilical cord-derived scaffolds

JING Hong-shu¹, PENG Yuan¹, WANG Zhen-yu¹, LI Wei-jian¹, YUAN Tian-jie², Zhang Hong-dan³, YAN He-xin^1,2, ZHAI Bo¹

1. Department of Interventional Oncology, Renji Hospital, Jiao Tong University School of Medicine, Shanghai 200127, China;
2. Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiao Tong University School of Medicine, Shanghai 200127, China;
3. Celliver Biotechnology Inc., Shanghai 201210, China

Received:2020-05-06 Online:2020-08-31 Published:2020-09-04
Contact: ZHAI Bo, Email: Zhaiboshi@sina.com

摘要/Abstract

摘要： 目的对脐带脱细胞支架(Human Decellularized Umbilical Cord Scaffolds, dHUCs)进行制备和表征,评价三维支架上培养小鼠肝前体样细胞(Rodent hepatocyte-derived liver progenitor-like cells, rHepLPCs)功能,提供一种新型组织工程化肝脏构建的新方案。方法两步灌肝法分离小鼠原代肝细胞,体外扩增并用Dil标记。基于脐带脱细胞支架材料的三维培养下rHepLPCs,通过测定成分检测、化学染色等方法表征材料,通过定量PCR、氨清除和尿素合成等方法进行rHepLPCs-3D的功能评价。结果经过脱细胞工艺处理后,脐带生物支架材料核酸残留量为(60.20±1.42)ng/mg(干重),核酸去除率达到90.5 %,基质成分含量丰富。小鼠肝前体样细胞体外扩增效果好,部分肝脏功能基因表达下降。支架培养2周后,ALB、G6PC和CPS1肝细胞功能基因的表达水平分别上调10.1±2.3、34.4±1.3、42.0±22.0倍。在氨代谢方面,2D培养的rHepLPCs氨清除量为(5.7±0.9)mg/(dL·10⁶细胞·d),尿素含量为(7.1±2.5) mg/(dL·10⁶细胞·d),而3D培养的rHepLPCs氨清除量为(26.7±4.5) mg/(dL·10⁶细胞·d),尿素量为(42.1±7.0)mg/(dL·10⁶细胞·d)。结论脐带脱细胞支架是理想的肝脏组织工程材料,3D培养rHepLPCs肝细胞功能明显提升,治疗以血氨升高为特征的急性肝衰竭具有明显优势。

关键词: 脐带脱细胞支架, 脱细胞技术, 小鼠肝前体样细胞, 转化与扩增培养基, 3D

Abstract: Objective This study aimed to fabricate and to characterize decellularized human umbilical cord-derived scaffolds (dHUCS), to evaluate the function of rodent hepatocyte-derived liver progenitor-like cells (rHepLPCs) cultured on 3-dimensional (3D) scaffolds, and to provide a novel liver tissue engineering protocol. Methods Hepatocytes were isolated from mice by a 2-step collagenase perfusion method, expanded in vitro and labeled with Dil. The dHUCS was characterized by detection of component, Masson staining and hematoxylin and eosin staining. The function of 3D-cultured rHepLPCs was analyzed by quantitative polymerase chain reaction and biochemical assays. Results After decellularization, the nucleic acid residue of umbilical cord-derived scaffolds was 60.20 ± 1.42 ng/mg (dry weight), and the removal rate of nucleic acids was 90.5%. Matrix component were rich. After 2-week culture, albumin, glucose-6-phosphatase and carbamoyl-phosphate synthetase 1 expression levels of hepatocytes were upregulated by 10.1 ± 2.3, 34.4 ± 1.3 and 42.0 ± 22.0 times, respectively. As for ammonia metabolism, the ammonia clearance and urea synthesis levels were 5.7 ± 0.9 mg/(dL·million cells·day) and 7.1 ± 2.5 mg/(dL·million cells·day) in 2-dimensional-cultured rHepLPCs, 26.7 ± 4.5 mg/(dL·million cells·day) and 42.1 ± 7.0 mg/(dL·million cells·day) in 3D-cultred rHepLPCs, respectively. Conclusion The dHUCS is an ideal liver tissue engineering material. The 3D cultured rHepLPCs could have better function, which have potential therapeutic effect on acute liver failure characterized by elevated serum ammonia.

Key words: Decellularized human umbilical cord-derived scaffold, Decellularization, Rodent hepatocyte-derived liver progenitor-like cell, Transformation and expansion medium, 3-dimensional

景宏舒, 彭媛, 王振宇, 李伟建, 袁天杰, 张洪丹, 鄢和新, 翟博. 脐带脱细胞支架培养鼠肝前体样细胞功能研究[J]. 肝脏, 2020, 25(8): 876-880.

JING Hong-shu, PENG Yuan, WANG Zhen-yu, LI Wei-jian, YUAN Tian-jie, Zhang Hong-dan, YAN He-xin, ZHAI Bo. Study on function of rHepLPCs cultured on decellularized human umbilical cord-derived scaffolds[J]. Chinese Hepatolgy, 2020, 25(8): 876-880.

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