共培养的大块组织工程肝构建及体内植入研究  

CONSTRUCTION OF LARGE BLOCK OF ENGINEERED LIVER TISSUE SEEDED WITH CO-CULTURED CELLS AND IN VIVO IMPLANTATION RESEARCH

作　　者：曲晓丽[1] 王敏[2] 贺健康[1] 刘亚雄[1] 周新民[2] 李涤尘[1] 靳忠民[1,3] 

机构地区：[1]西安交通大学机械制造系统工程国家重点实验室,西安710049 [2]第四军医大学西京消化病医院八科 [3]英国利兹大学医学与生物工程研究所

出　　处：《中国修复重建外科杂志》2014年第3期325-330,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划资助项目(2009AA043801);国家自然科学基金资助项目(51275387);中央高校基本科研业务费专项资金~~

摘　　要：目的选择大鼠成纤维细胞和原代肝细胞共培养于胶原水凝胶支架中,进行多细胞的大块组织工程肝构建及体内植入研究,以期构建长期存活的功能性肝组织。方法采用3-D打印技术制备硅胶模具,经成胶脱模制备胶原水凝胶支架。将大鼠肺成纤维细胞与原代肝细胞以0.4∶1比例种植于该支架中(B组),以种植同密度单纯原代肝细胞为对照(A组)。体外培养1、3、7、14、21 d,观察细胞形态并检测肝功能(白蛋白和尿素分泌量)。取24只SD大鼠,采用皮下注射四氯化碳方法制备肝硬化模型,将A、B组组织工程肝分别植入大鼠腹腔大网膜内(n=12),植入后3、7、14、21、28 d行HE、细胞角蛋白18(cytokeratin 18,CK18)及ALB染色观察。结果体外实验中,与A组相比,B组肝细胞呈多边形,细胞核大且圆,肝功能表达旺盛,并且在7 d时聚团生长最多,7 d后白蛋白及尿素分泌量显著增高(P<0.05)。体内实验中,B组肝细胞能存活至28 d,白蛋白和尿素分泌也显著增多,细胞团聚成条索形,形成了类正常肝脏组织。结论多细胞大块组织工程肝体内植入时形成了类正常肝脏组织,并长期存活,为构建结构和功能更完整的组织工程肝奠定了基础。Objective To construct large block of engineered liver tissue by co-culture of fibroblasts and hepatocytes on collagen hydrogels in vitro and do in vivo implantation research. Methods Silastic mould was prepared using three-dimensional printing technology. The collagen hydrogel scaffold was prepared by collagen hydrogel gel in the silicone mould and was removed. Sprague Dawley rat lung fibroblasts were co-cultured with primary hepatocytes at a ratio of 0.4 ∶ 1 on the collagen hydrogel scaffold to construct large block of engineered liver tissue in vitro （group B）, and primary hepatocytes cultured on the collagen hydrogel scaffold served as control group （group A）. The cell morphology was observed, and the liver function was tested at 1, 3, 7, 14, and 21 days after culture. The rat model （n=24） of hepatic cirrhosis was made by subcutaneous injection of carbon tetrachloride. And in vivo implantation study was carried in cirrhosis rat model. The phenotypic characteristics and functional expression of hepatocytes were evaluated at 3, 7, 14, 21, and 28 days after implantation. Results In vitro results indicated that hepatocytes in group B exhibited compact polyhedral cells with round nuclei and high expression of liver function. Moreover, cells aggregated to the most at 7 days. Album production and urea synthesis incresed significantly when compared with group A （P 〈 0.05）. In vivo results showed hepatocytes in group B survived for 28 days, and albumin production and urea synthesis were significantly increased. In addition, hepatocytes showed an aggregated distribution and cord-like structures, which was similar to normal liver tissue. Conclusion The large block of engineered liver tissue constructed by co-cultured cells can form tissue similar to normal liver tissue in vivo, and survive for a long time, laying foundations for building more complete engineered liver tissue in the future.

关 键 词：组织工程肝 3 D打印技术 胶原水凝胶支架 成纤维细胞 原代肝细胞 共培养 体内植入 大鼠 

分 类 号：R657.3[医药卫生—外科学]