以胶原凝胶为支架原位构建可注射型工程化肝

背景：为了解决肝移植供体的不足，最具潜力的肝组织工程技术悄然兴起，为肝组织的修复与功能重建开辟了新的前景。 目的：探索一种以新生大鼠肝细胞为种子细胞，以胶原凝胶为支架材料，可原位注射的工程化肝组织构建方法。 设计、时间及地点：以动物为观察对象的实验方法探索，于2007-03/2008-02在解放军总医院普通外科研究所完成。 材料：成年雌性SD大鼠及出生24 h以内的雄性新生SD大鼠。 方法：以胰酶消化法获取新生大鼠肝细胞，以PKH26标记后与液态胶原复合，采用注射方法植入大鼠肝脏。 主要观察指标：于肝细胞/胶原凝胶复合物植入当天及植入后第3，7天序贯取样、切片，分别行苏木精-伊红染色和白蛋白免疫组织化学染色后对“类肝组织”进行形态学观察，并观察植入细胞的示踪荧光。 结果：①以胶原为基质构建的工程化肝组织能方便地植入到肝脏。②荧光显微镜观察到移植区域主要由PKH26阳性细胞组成。③苏木精-伊红染色显示，移植区域肝细胞在胶原凝胶中可存活、生长。④免疫组织化学染色证实植入肝细胞能够表达白蛋白。 结论：以新生大鼠肝细胞/胶原凝胶为基础构建可原位注射的工程化肝组织是可行的，这种方法对于发展以组织工程为基础的原位肝脏重建具有良好的应用前景。

In situ construction of injectable engineered liver tissue using collagen gel as scaffolds

BACKGROUND: Respecting the donor of liver transplantation is insufficient, liver tissue engineering technology has gradually emerged and developed to establish a new way of liver tissue recovery and functional reconstruction. OBJECTIVE: To develop a novel method for constructing an engineered liver tissue in situ using collagen gel as a scaffold for neonatal rat hepatocytes. DESIGN, TIME AND SETTING: Animal experiments were carried out at the Institute of General Surgery of Chinese PLA General Hospital from March 2007 to February 2008. MATERIALS: Female adult SD rats and neonatal male SD rats within 24 hours were used. METHODS: The hepatocytes were isolated from neonatal rats by enzyme digestion method. After labled with PKH26, the seeding cells mixed with liquid collagen were transplanted into livers of adult rats by direct injection underneath the capsule. MAIN OUTCOME MEASURES: The hepatocytes/collagen gel specimens were harvested on days 0, 3 and 7 after implantation, respectively. Neotissue histology was evaluated by hematoxylin-eosin staining and differentiation was evaluated by immunohistology for albumin. Fluorescence microscopy of explanted liver was performed to identify donor cells. RESULTS: The collagen gel-immobilized hepatocytes were easily transplanted into liver by direct injection. The PKH26-positive cells were detected in the transplant area by fluorescence microscopy after transplantation. Hematoxylin-eosin staining showed the hepatocytes in the collagen survived and were integrated into the host liver after transplantation. Immunohistochemical staining demonstrated transplanted cells were positive for albumin. CONCLUSION: Direct intrahepatic injection of collagen gel-immobilized hepatocytes is technically feasible. Liver reconstruction in situ deserves potential use in tissue engineering field.