中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (16): 2541-2547.doi: 10.3969/j.issn.2095-4344.3149

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

三维胶原HepaRG微球的建立及其体外功能特征

黎   少，梁永康，高   毅，彭   青   

  1. 南方医科大学珠江医院肝胆二科，广东省广州市   510280
  • 收稿日期:2020-07-16 修回日期:2020-07-21 接受日期:2020-08-22 出版日期:2021-06-08 发布日期:2021-01-07
  • 通讯作者: 彭青，教授，南方医科大学珠江医院肝胆二科，广东省广州市 510280
  • 作者简介:黎少，女，1991年生，海南省陵水县人，汉族，南方医科大学毕业，硕士，研究实习员，主要从事功能肝脏细胞筛选与类器官构建研究。
  • 基金资助:
    国家重点研究计划(2018YFA0108200)，项目负责人：彭青；国家重点研究计划(2018YFC1106400)，项目负责人：高毅；国家自然科学基金项目(31972926)，项目负责人：高毅；广东省自然科学基金项目(2014A030312013)，项目负责人：高毅；广东省自然科学基金项目(2018A030313128)，项目负责人：彭青；广州市科技计划项目(201803010086)，项目负责人：彭青

Establishment and functional in vitro characteristics of three-dimensional collagen HepaRG microsphere

Li Shao, Liang Yongkang, Gao Yi, Peng Qing   

  1. Department of Hepatobiliary Surgery II, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Received:2020-07-16 Revised:2020-07-21 Accepted:2020-08-22 Online:2021-06-08 Published:2021-01-07
  • Contact: Peng Qing, Professor, Department of Hepatobiliary Surgery II, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • About author:Li Shao, Master, Department of Hepatobiliary Surgery II, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Supported by:
    the National Key Research & Development Program of China, No. 2018YFA0108200 (to PQ); the National Key Research & Development Program of China, No. 2018YFC1106400 (to GY); the National Natural Science Foundation of China, No. 31972926 (to GY); the National Science Foundation of Guangdong Province, No. 2014A030312013 (to GY); the Natural Science Foundation of Guangdong Province, China, No. 2018A030313128 (to PQ); the Science and Technology Program of Guangzhou, No. 201803010086 (to PQ)

PDF

58

摘要：

文题释义：
胶原微球：将鼠尾I型胶原蛋白与细胞充分混合后形成细胞-胶原水凝胶三维微球，其形态为规整的球形结构，微球内可观察到细胞均匀分布，具有较好的细胞-基质交互作用，同时物质交换及细胞形态、增殖、生长和功能表达等都不受限制。
HepaRG细胞：是一种人源肝祖细胞，可进一步分化成为成熟肝细胞或胆管细胞，细胞具有较高的药物代谢相关酶活性、尿素合成、转运蛋白等肝功能表达，这些特征与人原代肝细胞(PHH)较为接近，目前被应用于药物代谢、组织工程等方面的研究。

背景：近年来的一些研究显示，HepaRG细胞三维培养模型可较好地模仿体内微环境，与二维培养相比显示出更好的肝分化和功能情况。
目的：选择HepaRG人源肝祖细胞制备三维胶原微球，评价胶原微球内细胞适应性培养和功能表达情况。
方法：以胶原蛋白作为基质材料，选择HepaRG和HepG2细胞分别建立三维胶原细胞微球模型，形成稳定的细胞球体，以HepaRG普通微球、HepG2普通微球、HepaRG二维培养、HepG2细胞二维培养为对照。培养1，6，12 d后，死活染色法观察细胞存活情况；培养1，6，12，16 d后，检测各组上清尿素合成与CYP3A4分泌量；培养12 d后，采用qPCR检测CYP3A4、CYP1A2、UGT1A1、CPS1 mRNA相对表达，Western Blot检测肝细胞标志物白蛋白和CYP3A4蛋白表达水平。
结果与结论：①连续培养12 d的活死染色显示，三维胶原微球内的活细胞数量较多，死细胞较少，具有较高的细胞活率，并且HepaRG三维胶原微球内的细胞呈现出多个细胞簇紧密连接的交联结构；②HepaRG三维胶原微球培养1，6，12 d后的尿素含量高于HepaRG二维培养(P < 0.05)，HepG2三维胶原微球培养1，6，12，16 d后的尿素含量高于HepG2二维培养(P < 0.05)，HepaRG三维胶原微球培养1，6，12 d后的CYP3A4分泌量高于HepaRG二维培养(P < 0.05)，HepG2三维胶原微球培养6，12 d后的CYP3A4分泌量高于HepG2二维培养(P < 0.05)；③HepaRG三维胶原微球内的CYP3A4、CYP1A2、UGT1A1和CPS1 mRNA相对表达高于HepaRG二维细胞(P < 0.05)，HepG2三维胶原微球内的CYP1A2相对表达高于HepG2二维培养(P < 0.05)；④HepaRG三维胶原微球内的白蛋白、CYP3A4蛋白表达水平高于HepG2三维胶原微球、普通微球、二维培养(P < 0.05)；⑤结果表明，HepaRG三维胶原微球具有高表达的肝功能水平，可为体外药物代谢评价、组织工程等应用提供研究参考。

关键词: 材料, HepaRG细胞, 三维培养, 胶原微球, 细胞表型, 细胞色素P450酶系, 尿素合成, 基因表达

Abstract: BACKGROUND: Some recent studies have shown that the three-dimensional (3D) model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture. 
OBJECTIVE: HepaRG was selected to prepare 3D collagen microspheres, and the adaptive culture and functional expression of cells in the collagen microspheres were evaluated. 
METHODS: Collagen hydrogel was used as the scaffold for 3D HepaRG and HepG2 microspheres. Stable cell spheres were formed. HepaRG microspheres, HepG2 microspheres, HepaRG two-dimensional culture and HepG2 two-dimensional culture were used as controls. At 1, 6, and 12 days of culture, cell survival was detected by the Live/Dead assay staining. After 1, 6, 12, and 16 days of culture, the urea synthesis and CYP3A4 secretion of the supernatant were detected in each group. After 12 days of culture, relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA was detected by qPCR. The expression levels of hepatocyte marker albumin and CYP3A4 protein were determined using western blot assay. 
RESULTS AND CONCLUSION: (1) In 12 days of culture, Live/Dead assay staining showed that the cell viability in the 3D collagen microsphere was well-maintained and the amount of central necrotic cells was small, with high cell viability. In the 3D collagen microsphere, especially HepaRG cells, multiple cellular clusters formed and adjacent clusters were connected closely, which created a cross-linked structure. (2) After 1, 6, and 12 days of culture, the urea content of HepaRG 3D collagen microspheres was higher than that of HepaRG two-dimensional culture (P < 0.05). After 1, 6, 12, and 16 days of culture, the urea content of HepG2 3D collagen microspheres was higher than that of HepG2 two-dimensional culture (P < 0.05). After 1, 6, and 12 days of culture, the secretion of CYP3A4 in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional culture (P < 0.05). After 6 and 12 days of culture, the secretion of CYP3A4 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (3) The relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional cells (P < 0.05), and the relative expression of CYP1A2 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (4) The expression levels of albumin and CYP3A4 protein in HepaRG 3D collagen microspheres were higher than those of HepG2 3D collagen microspheres, ordinary microspheres, and two-dimensional culture (P < 0.05). (5) These results indicated the high-level expression of hepatocyte functions in 3D collagen HepaRG microsphere, which could be taken as a reference in drug metabolism evaluation in vitro and tissue engineering application. 

Key words: material, HepaRG cell, three-dimensional culture, collagen microsphere, cellular phenotype, cytochrome P450 system, urea secretion, gene expression

中图分类号: