胚胎干细胞来源的肝细胞对急性肝功能衰竭小鼠的移植治疗作用

目的观察胚胎干细胞（ESC）来源的肝细胞对急性肝功能衰竭（FHF）小鼠的移植治疗方法和作用。方法选用小鼠D3-KS细胞，利用转化生长因子、成纤维细胞生长因子、肝细胞生长因子等进行肝细胞方向诱导分化；用反转录聚合酶链反应、免疫细胞化学等方法检测肝细胞标记物甲胎蛋白（AFP）、白蛋白（ALB）和葡萄糖6磷酸酶（G6P）等的表达；观察阳性细胞的空间分布规律，用细胞铲分选阳性区域细胞并将其移植入同系（129系）小鼠肝脏内，利用四氯化碳诱发FHF，然后观察小鼠生存时间、肝功能指标和移植细胞生长状态等情况。结果（1）AFP、白蛋白和G6P等最早于第3天开始表达并逐渐增强。（2）移植组平均生存时间（60h）与对照组（22h）差异有统计学意义（P〈0．05）；移植组肝功能指标均较对照组明显改善（P〈0．05）。（3）移植细胞表达ALB，移植部位未发现肿瘤形成。结论ESC来源的肝细胞可表达成熟肝细胞功能，移植后能对小鼠FHF起到较好的治疗作用。

Curative effects of transplantation of hepatocytes differentiated from embryonic stem cells on treatment of fulminant hepatic failure: experiment with mice

Objective To investigate the curative effects of transplantation of hepatocytes differentiated from embryonic stem cells (ESCs) on treatment of fulminant hepatic failure (FHF). Methods Mouse ESCs transfected with green fluorescent protein were cultured. RT-PCR was used to detect the mRNA expression of alpha-fetoprotein (AFP), tranthyretin (TTR), hepatic nuclear factor (HNF), albumin (ALB), glucose-6-phophate (G6P), and tyrosine aminotransferase (TAT) at different time points. Imunocytochemistry (ICC) was used to detect the expression of AFP, ALB, cytokeratin 8 (CK8), and CK11 in the cells. The morphology and distribution of the cells were observed with microscope. Forty mice were randomly divided into 2 equal groups: ESC transplantation group, undergoing transplantation of ICC positive ESCs (hepatocytes) into the hepatic capsule, and control group, undergoing injection of normal saline in the hepatic capsule. Twenty-four hours later carbon tetrachloride was injected intra-peritoneally to induce FHF. The survival status of the mice was observed. Twenty-four hours later venous blood samples were collected to examine the levels of total bilirubin (TB), alanine aminotransferase (ALT), ALB, blood sugar (BS), and prothrombin time (PT). After the death of the mice, their livers were taken out to undergo microscopy and immunohistochemistry to observe the structure of liver tissue, growth of tumor, and expression of ALB. Results RT-PCR showed that the mRNA expression of AFP, TTR, HNF, ALB, G6P, and TAT emerged since days 3, 3, 5, 9, and 11 respectively and then gradually increased till day 19. ICC showed that the EB cells began to express AFP since day 7, to express CK8 and CK11 since day 9, and to express ALB since day 11. The ICC-positive cells were consistent with the mouse hepatocytes morphologically and were distributed only in the central and marginal areas of the EB cell community. Injected with carbon tetrachloride, the mice showed manifestations of FHF. However, the symptoms of central nervous system emerged later in the mice implanted with the hepatocytes in comparison with the control mice; 8 of the 20 mice in the transplantation group showed ascites in comparison with 14 in the control group. The mean survival time of the transplantation group was 62 hours, significantly longer than that of the control group (23 hours, P<0.05). Compare with the normal mice, the FHF mice in both groups showed higher ALT and TB and lower ALB and BS (all P<0.01), however, the levels of ALT and TB were lower, the level of BS was higher, and PT was shorter significantly in the transplantation group than in the control group (all P<0.05). Pathology showed that no tumor formation was found in both groups and that the transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed ALB. Conclusion Hepatocytes originating from ESCs exercise the functions of normal hepatocytes. Transplantation of hepatocytes differentiated from ESCs is able to improve the life quality and lengthen the survival time of FHF mice.