大鼠肝细胞在肝脏脱细胞支架内的体外三维循环灌注培养  

Three-dimensional circulation perfusion culture of hepatocytes in the liver decellularizedscaffold

作　　者：王雷[1] 周鹏成 朱沙俊[1] 王尧[2] 范向军[2] 朱铭岩[2] 王志伟[2] 钱海鑫[1] 

机构地区：[1]苏州大学附属第一医院普通外科,江苏苏州215006 [2]南通大学附属医院普通外科

出　　处：《中华医学杂志》2017年第4期265-269,共5页National Medical Journal of China

基　　金：国家自然科学基金（81471801）

摘　　要：目的制备大鼠肝脏脱细胞支架，将肝细胞再植于脱细胞支架并进行体外循环灌注培养，为肝脏脱细胞支架应用于肝脏组织工程学提供实验基础依据。方法采用灌注法制备大鼠肝脏脱细胞支架，HE、苦味酸一天狼星染色、免疫组织化学检测脱细胞支架结构及成分，扫描电镜显示脱细胞支架超微结构，DNA含量检测脱细胞效果；构建循环灌注培养体系；采用经门静脉途径和包膜直接注射这两种方法将肝细胞种植于脱细胞支架内，行体外循环灌注培养，检测支架内肝细胞的代谢功能，培养完成后行HE、ALB免疫荧光、扫描电镜，观察细胞在支架内生长情况。结果采用灌注法获得大鼠肝脏脱细胞支架，组织学染色未见细胞核成分，同时可见细胞外基质成分保留；免疫荧光显示Ⅰ型胶原蛋白保留；扫描电镜显示细胞外基质呈网状超微结构；DNA含量为（47．5±18．1）ng／mg；构建出由蠕动泵、氧合器、培养瓶、输送管道组成的循环灌注培养体系，包膜直接注射法种植成功率优于经门静脉途径，大鼠肝细胞在支架内合成、代谢功能良好，HE、免疫荧光、扫描电镜显示肝细胞在脱细胞支架的细胞外基质三维环境中定植，生长状态良好。结论采用循环灌注法获得的大鼠肝脏脱细胞支架具有良好的理化性质，循环灌注培养体系结合肝脏脱细胞支架可以为肝细胞提供良好的三维生长环境。Objective The intact rat liver decellularized scaffolds were preparedand repopulated hepatocytes by continuous perfusion technology. Toprovideexperimental support for the application of decellularized liver scaffolds in liver engineering. Method Decellularized liver scaffolds were obtained by perfusing method. The composition and structure was examined by HE, Masson, Sirius red stain and immunofluorescence. The ultrastructure was examined by scanning electron microscope （SEM）. DNA content was used to confirm the effect of decellularization. The circulation perfusion device was established. Hepatocytes were recellularized into the scaffolds by muhiposition parenchymal injection method and infusion method,thenthe scaffolds were cultured in the circulation perfusion device in vitro. After cultivation, HE staining, immunofluorescence and SEM were conducted to observe the growth situation of hepatocytes in the scaffolds. Results The rat decellularized liver scaffolds were successfully obtained by perfusion method. Histological staining demonstrated the remove of cellular component and the reservation of extraeellular cellmatrix. Immunofluorescence staining demonstrated the retention of collagen I. SEM showed that the ultrastructure of the extracellular cell matrix presented thereticular structure. DNA content of the scaffolds was 47.5 _＋ 18. 1 ng/mg. The circulation perfusion device was composed of a peristaltic pump, oxygenator, chamber and the convey tubes. The multipositional parenchymal injection method resulted in a better engraftment rate. HE staining, immunofluorescence and SEM revealed that the growth and function of hepatocytes were goodin the scaffold. Conclusion The decellularized rat liver scaffolds have favorable biochemical properties. The liver decellularized scaffolds applied with the circulation perfusion device could provide a well 3D plat form for culture of hepatocytes.

关 键 词：肝脏 组织工程 组织支架 灌注 

分 类 号：R575[医药卫生—消化系统]