脱细胞三维肝脏组织工程支架的初步研究——脱细胞和超微结构评价

Preliminary study of three-dimensional liver tissue specific matrix scaffold-decellularization and ultrastructure evaluation

目的 通过不同脱细胞方法制备猪肝源性三维多孔特异性细胞外基质（Extracellular Matrix,ECM）,为人原代肝细胞体外长期增殖、组织构建和功能保持提供实验基础.方法 使用六种脱细胞程序处理猪肝脏薄片组织,采用定性和定量分析对脱细胞程度和ECM结构进行观察.结果 脱细胞的肝脏ECM在整个去细胞过程中都保持了它们的原始外形.HE染色显示脱细胞基质内细胞核及细胞成分消失,证明六种脱细胞方法均可彻底完成薄片猪肝脏组织的脱细胞化.DNA含量测定表明细胞成分去除率为91％ ～97％.扫描电镜观察显示六种脱细胞方法处理的肝脏ECM孔隙大小和超微结构有明显差异.结论 本研究采用六种脱细胞/氧化方法成功研制了具有多孔超微结构的三维脱细胞肝脏细胞外基质,为创建新型人肝细胞体外三维培养模型奠定了基础.

Objective To provide an experimental basis for long-term in vitro proliferation,tissue construction and function maintaining of human primary hepatocytes by preparing three-dimensional （3D） porous specific extracellular matrix （ECM） derived from porcine liver with different decellularizing methods.Methods The porcine liver tissue slices were treated using six different decellularization/oxidation ways,and the decellularization extent and ECM structure were evaluated through qualitative and quantitative analysis.Results The decellularized liver-ECM could maintain the original shape during the whole process of the decellularization.HE staining showed that the cellular components and nucleus disappeared in the decellularized ECM,indicating that all of these six decellularization methods can completely decellularize the porcine liver tissue slices.Determination of DNA content showed that 91％ ～ 97％ of cell components have been removed.Electron microscopy scanning observed that the pore sizes and ultrastructures of the liver ECMs under six decellularization treatments were significantly different.Conclsion The six decellularization/oxidation methods evaluated in this study could successfully create a 3D specific decellularized liver ECM with porous ultrastructure,which further lays basis for the development of a novel in vitro 3D culture model for human hepatocytes.