三维胶原HepaRG微球的建立及其体外功能特征  

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

作　　者：黎少 梁永康 高毅[1] 彭青[1] Li Shao;Liang Yongkang;Gao Yi;Peng Qing(Department of Hepatobiliary Surgery II,Zhujiang Hospital of Southern Medical University,Guangzhou 510280,Guangdong Province,China)

机构地区：[1]南方医科大学珠江医院肝胆二科,广东省广州市510280

出　　处：《中国组织工程研究》2021年第16期2541-2547,共7页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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1,6,12,16d后,检测各组上清尿素合成与CYP3A4分泌量;培养12d后,采用qPCR检测CYP3A4、CYP1A2、UGT1A1、CPS1mRNA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Hepa RG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;②Hepa RG三维胶原微球培养1,6,12 d后的尿素含量高于Hepa RG二维培养(P<0.05),Hep G2三维胶原微球培养1,6,12,16 d后的尿素含量高于Hep G2二维培养(P<0.05),Hepa RG三维胶原微球培养1,6,12 d后的CYP3A4分泌量高于Hepa RG二维培养(P<0.05),Hep G2三维胶原微球培养6,12 d后的CYP3A4分泌量高于Hep G2二维培养(P<0.05);③Hepa RG三维胶原微球内的CYP3A4、CYP1A2、UGT1A1和CPS1 mRNA相对表达高于Hepa RG二维细胞(P<0.05),Hep G2三维胶原微球内的CYP1A2相对表达高于Hep G2二维培养(P<0.05);④Hepa RG三维胶原微球内的白蛋白、CYP3A4蛋白表达水平高于Hep G2三维胶原微球、普通微球、二维培养(P<0.05);⑤结果表明,Hepa RG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。BACKGROUND:Some recent studies have shown that the three-dimensional(3 D)model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture.OBJECTIVE:Hepa RG was selected to prepare 3 D 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 3 D 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 CYP3 A4 secretion of the supernatant were detected in each group.After 12 days of culture,relative expression of CYP3 A4,CYP1 A2,UGT1 A1,and CPS1 mRNA was detected by qPCR.The expression levels of hepatocyte marker albumin and CYP3 A4 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 3 D collagen microsphere was wellmaintained and the amount of central necrotic cells was small,with high cell viability.In the 3 D 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 3 D 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 HepG23 D 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 CYP3 A4 in HepaRG 3 D collagen microspheres was higher than that in HepaRG two-dimensional culture(P<0.05).After 6 and 12 days of culture,the secretion of CYP3 A4 in HepG23 D collagen microspheres was higher tha

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 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]