胚胎干细胞源性肝干细胞肝内移植对宿主肝功能的影响及安全性评估(英文)

Effect of intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells on host hepatic function and its safety evaluation

背景：体外诱导胚胎干细胞分化为肝细胞已有不少成功的报道，但其体内移植后能否有效整合入宿主肝板、在肝内能否进一步生长分化并表达肝细胞功能以及成瘤的风险等情况目前还不清楚。目的：应用治疗性肝再生模型进行胚胎干细胞源性肝干细胞肝内移植．观察其在肝组织替代、体内的生长分化及成瘤性情况。设计：随机对照动物实验。单位：中山大学附属第二医院小儿外科。材料：选用BALB／c小鼠24只为受体，鼠龄6～8周，体质量20～352，雌雄不拘购自广州市实验动物中心。实验所用胚胎干细胞源性肝干细胞由作者所在课题组诱导胚胎干细胞分化而成。小鼠胚胎干细胞株E14由本院干细胞中心提供。方法：实验于2006-07／2007-06在中山大学附属第二医院干细胞研究中心完成。将24只小鼠随机分为2组：肝再生模型＋干细胞移植组和肝切除＋干细胞移植组，每组12只。前组分两次按50mg／kg剂量腹腔内注射倒千里光碱（retrorsine），间隔2周，第2次注射4周后行70％肝部分切除制造肝损伤；然后经门静脉分别移植1×10^5羟基荧光素乙酰乙酸（CFDA-SE）荧光标记的细胞入小鼠肝内进行胚胎干细胞源性肝干细胞移植。后组在行70％肝部分切除制造肝损伤模型后进行胚胎干细胞源性肝干细胞移植。主要观察指标：荧光显微镜下观察移植细胞组受体鼠肝脏内分布、整合与体内生长分化情况。2周后行白蛋白荧光免疫组化（双荧光染色）、血清白蛋白水平检测其功能状况。将胚胎干细胞源性肝干细胞注入治疗性肝再生小鼠肝内，将未分化的胚胎干细胞移植入小鼠腋区皮下作为对照，观察胚胎干细胞源性肝干细胞体内成瘤情况。结果：①肝干细胞在受体鼠肝内生长情况：CFDASE标记的胚胎干细胞源性肝干细胞肝内移植1周，受体小鼠肝实质内可见散在绿色荧光分布。2周后，肝实质内绿色荧光分布区域明显扩大，且可见类似肝索样结构排列。②肝功能：共焦白蛋白荧光免疫组化（双荧光染色）结果表明，受体小鼠肝组织内可见标记细胞表达白蛋白阳性信号（呈黄色荧光），肝再生模型＋干细胞移植组和肝切除＋干细胞移植组血清白蛋白水平则无明显差异（P〉0．05）。③肝干细胞移植安全性：6周内未见畸胎瘤形成，而将未分化的胚胎干细胞移植入小鼠腋区皮下6周后则可见畸胎瘤形成。结论：胚胎干细胞源性肝干细胞移植入治疗性肝再生模型小鼠肝内后可有效在肝内能进一步生长分化并部分表达肝细胞功能：且此移植安全性较好。

BACKGROUND： In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present. OBJECTIVE： To study the intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation models, and to investigate the liver tissue replacement, growth and differentiation in vivo, and neoplastic formation. DESIGN： Randomized controlled animal study. SETTING： Department of Pediatric Surgery, the Second Hospital affiliated to Sun Yat-sen University. MATERIALS： Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, were provided by Guangzhou Experimental Animal Center. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. El4 was provided by Stem cell Center of our hospital. METHODS： This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four mice were randomly divided into a liver repopulation model ＋ stem cell transplantation group （group A） and a liver resection ＋ stem cell transplantation group （group B）, with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1 × 10^5 carboxy fluoresce in diacetate succinimidyl ester （CFDA-SE）, were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver. MAIN OUTCOME MEASURES： The distribution, incorporation, and proliferation of transplanted cells were observed under fluorescent microscopy. Two weeks later, hepatic function was stained with albumin fluorescence immunoassay （double fluorescence staining） and assayed by level of serum albumin. Embryonic stem cells-derived hepatic stem cells were poured into liver of remedial liver regeneration mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells. RESULTS： ① Growth of hepatic stem cells in recipient mice： One week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, some scattered region was green under fluorescent microscopy. The area of green region increased apparently in, 2 weeks, and cord-like structure could be observed. ② Liver function： Immunofluorescent staining of albumin （double fluorescence staining） demonstrated that labeled cells expressed positive albumin （yellow fluorescence） in liver tissue of recipient mice, but there was not significant difference in serum albumin level between group A and group B （P 〉 0.05）. ③ Reliability of hepatic stem cell transplantation： Teratoma did not form over 6 months; however, transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks. CONCLUSION： The transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively and further grow and differentiate, or even partially express hepatocyte function; in particular, the transplantation is safe.