移植肝细胞与胶原水凝胶单元皮下创建工程化肝组织

Transplanted hepatocytes and collagen hydrogel units subcutaneously establish engineered hepatic tissue

背景:胶原水凝胶可为肝细胞生长和组织重建提供良好的基质支持,并且以胶原为基质构建的工程化组织易于合并生长,形成融合组织。目的:尝试将肝细胞/胶原水凝胶复合物打散成小块肝单元,堆积在皮下腔中,提高工程化肝组织厚度。方法:将新鲜分离的大鼠肝细胞与胶原水凝胶复合,构建肝细胞/胶原水凝胶复合物,待固化后将其打散成小块肝单元,以未打散的肝细胞/胶原水凝胶复合物作为对照。取6只SD大鼠,其中3只切除2/3肝脏,诱导肝再生,于双侧腹股沟皮下腔中分别植入打散与未打散的肝细胞/胶原水凝胶复合物;另3只于双侧腹股沟皮下腔中分别植入打散与未打散的肝细胞/胶原水凝胶复合物。植入后7 d取材,采用苏木精-伊红染色、免疫组化染色、印度墨水灌注等方法对工程化肝组织形成情况进行评价。结果与结论:两组移植物均在皮下腔中形成了血管工程化肝组织,但小块肝单元相互融合,形成了大块血管工程化肝组织,肝组织厚度可达4 mm;整块植入的肝移植物只形成小块肝组织,其厚度只有几层细胞。免疫组织化学染色证实,血管工程化肝组织中的肝细胞具有天然肝细胞的特征及功能。肝部分切除实验表明,工程化肝组织具有对肝部分切除再生刺激产生反应的能力。结果表明将肝细胞/胶原水凝胶移植物打散成小块单元,堆积在皮下腔中,可提高工程化肝组织的厚度。

BACKGROUND： Collagen hydrogel provides good matrix support for hepatocyte growth and tissue reconstruction,and the collagen-based engineered tissue is easy to merge the growth and form integrated tissue. OBJECTIVE： To improve the thickness of engineered hepatic tissue by dissociating hepatocytes/collagen hydrogel composite into small hepatic units that accumulate in the subcutaneous cavity. METHODS： Freshly isolated hepatocytes from rats were mixed with collagen hydrogel to establish hepatocytes/collagen hydrogel composite. The hepatocytes/collagen hydrogel composite was dissociated into small hepatic units after being cured. The undissociated hepatocytes/collagen hydrogel composite was taken as a control. Six Spraque-Dawley rats were enrolled. Three of them were subjected to a two-thirds partial hepatectomy to induce liver regeneration. Dissociated and undissociated hepatocytes/collagen hydrogel composites were implanted into the bilateral inguinal subcutaneous cavity. Dissociated and undissociated hepatocytes/collagen hydrogel composites were implanted into the bilateral inguinal subcutaneous cavity of the other three rats. At the 7th day after transplantation,engineered hepatic tissue formation was evaluated using hematoxylin-eosin staining,immunohistochemical staining and India ink perfusion methods. RESULTS AND CONCLUSION： The grafts in these two groups all formed vascular engineered hepatic tissue inthe subcutaneous cavity,but after the small hepatic units merged,a large piece of vascular engineered hepatic tissue formed. The hepatic tissue thickness was up to 4 mm. The whole piece of implanted liver grafts only formed small pieces of hepatic tissues,with only several layers of cells. Immunohistochemistry staining confirmed that the hepatocytes in vascular engineered hepatic tissue had the characteristics and functions of natural hepatocytes. Partial hepatectomy experiment showed that engineered hepatic tissue had the ability to respond to regenerative stimulus of partial hepatectomy. These results show that dissociating the hepatocytes/collagen hydrogel grafts into small units that accumlate in the subcutaneous cavity can increase the thickness of the engineered hepatic tissue.