肝细胞、肝窦内皮细胞分离培养及肝细胞支架的制备

目的：探究大鼠肝细胞、肝窦内皮细胞的分离培养方法及肝去细胞支架的制备方法。方法：用胶原酶IV消化肝脏组织后经洗涤、过滤、离心等步骤分离出大鼠肝细胞，然后置于培养基内进行纯化、培养后行细胞免疫组织化学检测；通过Percoll密度梯度离心法分离出肝窦内皮细胞置于培养基内纯化、培养后行免疫荧光检测；肝组织经循环灌注去细胞处理制备成大鼠肝细胞去细胞支架后经扫描电镜证实。结果：分离出的大鼠肝细胞在显微镜下呈光亮圆形，饱满，胞核清晰。肝细胞细胞角蛋白18免疫组织化学染色后见胞浆内存在棕黄色颗粒。肝窦内皮细胞呈梭形，细胞核位于中央。内皮素-1免疫荧光染色后细胞表面呈红色信号。制备成的大鼠肝脏去细胞支架在扫描电镜下显示细胞外基质保留，无细胞核及细胞质成分。结论：成功分离出了肝细胞和肝窦内皮细胞并成功制备了肝去细胞支架。

Isolation and culture of hepatocytes and sinusoidal endothelial cells and preparation of hepatocyte scaffolds

Objective: To explore the methods of isolation and culture of rat hepatocytes and sinusoidal endothelial cells and preparation of hepatic acellular scaffolds. Methods: Rat hepatocytes isolated from liver tissues were digested by collagenase IV and purified in culture medium after washing, filtration and centrifugation. Cell immunohistochemistry was used to detect the cells after culture. Hepatic sinusoidal endothelial cells were isolated by Percoll density gradient centrifugation and purified in culture medium before they were detected by immunofluorescence. The scaffolds of rat hepatocyte acellular scaffolds were prepared by circulating perfusion and acellular treatment. The scaffolds were confirmed by scanning electron microscopy. Results: The isolated rat hepatocytes were round, full and clear under microscope. Immunohistochemical staining of cytokeratin 18 in hepatocytes revealed brown granules in the cytoplasm. Thehepatic sinusoidal endothelial cells were spindle-shaped and the nucleus was located in the center. Endothelin-1 immunofluorescence staining showed red signal on the cell surface. The scanning electron microscopy of the rat liver decellularized scaffold showed that the extracellular matrix was retained, and no nuclear and cytoplasmic components were found. Conclusion: Hepatocytes and sinusoidal endothelial cells are successfully isolated and hepatic acellular scaffolds were prepared.