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背景:目前针对肝癌是起源于成熟肝细胞的去分化还是肝干细胞的成熟受阻这一问题仍存在争议。目的:观察胚胎肝干细胞癌变的可能性。设计、时间和地点:随机对照动物实验,于2006-01/12在中山大学动物实验中心完成。材料:6～8周龄BALB/c雌性小鼠70只,体质量20～25g,随机分为3组:空白对照组10只、模型组30只、实验组30只。另取孕14d胎鼠用于制备胚胎肝细胞。方法:3组小鼠麻醉后均行2/3肝切除。模型组诱导肝癌,在饮水中加入100μg/L二乙基亚硝胺,连续饮用12周。实验组在诱导肝癌的基础上行肝干细胞移植,即分离的胚胎肝细胞经肠系膜静脉注射到肝脏,每只小鼠移植1×106个细胞。空白对照组仅行肝干细胞移植,同时饮正常水。6个月后处死小鼠,制备肝脏标本,取肝癌结节做连续切片进行指标检测。主要观察指标:采用免疫组化或免疫荧光方法检测Y染色体性别决定区域蛋白、甲胎蛋白、胎盘型谷胱苷肽转移酶或细胞角蛋白19的表达,以分析肝癌的细胞来源和特征。结果:6个月后,实验组有8只小鼠成功诱癌,其中7只为肝细胞性肝癌,1只为胆管细胞性肝癌;模型组有7只小鼠成功诱癌,其中6只为肝细胞性肝癌,1只为胆管细胞性肝癌;两组诱癌率、肿瘤病理类型比较均无明显差异(P>0.05)。实验组17.1%的肝细胞癌结节Y染色体性别决定区域蛋白染色阳性,胆管细胞癌结节Y染色体性别决定区域蛋白染色均为阴性。肝细胞癌结节表达甲胎蛋白和谷胱苷肽转移酶,而不表达细胞角蛋白19;胆管细胞癌结节不表达甲胎蛋白和谷胱苷肽转移酶,而表达角蛋白19。结论:在诱导肝癌过程中移植的肝干细胞有癌变的潜能,并保持了胚胎肝干细胞未成熟的特征,仍表达谷胱苷肽转移酶和甲胎蛋白。
BACKGROUND: Currently there is still controversy over whether hepatocellular carcinoma (HCC) is derived from dedifferentiation of mature hepatocytes or from maturation of hepatic stem cells. Objective: To observe the possibility of embryonic hepatic stem cell carcinogenesis. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Animal Experimental Center of Sun Yat-sen University from January to December 2006. MATERIALS: Seventy-six BALB / c female mice aged 6-8 weeks were randomly divided into 3 groups: control group (n = 10), model group (n = 30) and experimental group (n = 30). Another fetus 14d pregnant embryos for the preparation of embryonic liver cells. Methods: Three groups of mice underwent 2/3 hepatectomy after anesthesia. Model group induced liver cancer, adding 100μg / L diethylnitrosamine in drinking water, continuous drinking for 12 weeks. In the experimental group, liver stem cell transplantation was performed on the basis of inducing liver cancer, that is, the isolated embryonic liver cells were injected into the liver through the mesenteric vein, and each mouse transplanted 1 × 10 6 cells. Blank control group only hepatic stem cell transplantation, while drinking normal water. Mice were sacrificed 6 months later, liver specimens were prepared, and serial sections of liver cancer nodules were taken for index detection. MAIN OUTCOME MEASURES: Immunohistochemical or immunofluorescent staining was used to detect the sex determination of Y chromosome protein, alpha-fetoprotein, placental glutathione transferase or cytokeratin 19 in order to analyze the origin and characteristics of hepatocellular carcinoma cells. Results: After six months, eight mice in the experimental group were successfully induced cancer, of which seven were hepatocellular carcinoma and one was cholangiocarcinoma. Seven mice in the model group were successfully induced cancer, of which six were Hepatocellular carcinoma, 1 cholangiocarcinoma only. There was no significant difference in tumor-inducing rate and tumor pathological type between the two groups (P> 0.05). In experimental group, 17.1% of hepatocellular carcinoma nodules y chromosome sex determination region protein positive staining, cholangiocarcinoma nodules y chromosome sex determination region protein staining were negative. Hepatocellular carcinoma nodules express alpha-fetoprotein and glutathione transferase but not cytokeratin 19; cholangiocarcinoma nodules do not express alpha-fetoprotein and glutathione transferase but express keratin 19. Conclusion: The hepatic stem cells transplanted in the process of inducing hepatocarcinoma have the potential of carcinogenesis and maintain the immature characteristics of embryonic liver stem cells, and still express glutathione transferase and alpha-fetoprotein.