胚胎肝干细胞癌变实验

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20～25 g,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14 d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100 μg/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.

Potential of carcinogenesis from hepatic progenitor cells in hepatocellular carcinoma

BACKGROUND: Whether hepatocellular carcinomas (HCC) arise from dedifferentiation of mature hepatocytes or maturation arrest of hepatic stem calls is controversial. OBJECTIVE: To investigate the malignant potential of hepatic progenitor cells derived from the fetal liver in a mouse model of chemical hepatocarcinogenesis. DESIGN, TIME AND SETTING: The randomized controlled animal experiment was performed at the Animal Experimental Center of Sun Yat-sen University from January to December 2006. MATERIALS: A total of 70 female BALB/c mice aged 6-8 weeks and weighing 20-25 g were used. These mice were randomly assigned to a normal control group (n=10), a model group (n=30) and an experimental group (n=30). Hepatic progenitor were obtained from embryonic day (ED) 14 mice.METHODS: Partial hepatectomy was undertaken using the standard method for a two-thirds resection. Thirty mice in the model group were continuously administered 100 μg/L diethylnitrosamine in the drinking water for 12 weeks. Thirty mice in the experimental group received hepatic progenitor cells and diethylnitrosamine, 1× 106 cells in each mouse. Ten mice in the normal control group were given hepatic progenitor cells and non-supplemented drinking water. Animals were sacrificed at six months to prepare liver samples. Hepatoma nodules were used to make serial sections for determination. MAIN OUTCOME MEASURES: Immunohistochemistry or immunofluorescence was performed for sex-determining region for Y chromosome (SRY), alpha-fetoprotein, placental form of glutathione-S-transferase (GST-P) or cytokeratin 19 in serial sections of the liver and tumor nodules to analyze cell source and characteristics of HCC. RESULTS: Six months later, seven mice developed hepatocellular carcinoma (HCC) and one mouse developed chotangiocellular carcinoma (CCC) in experiment group. Six affected HCC and one affected CCC. There were no significant differences in carcinoma occurrence rate and tumor pathological types between both groups (P > 0.05). About 17.1% of HCCs stained positively for SRY and none of CCCs was positive for SRY in the experimental group. Most of the SRY-positive HCCs expressed GST-P and alpha-fetoprotein, but did not expressed cytokeratin 19. All of CCCs expressed cytokeratin 19 in the cytoplasm, but did not expressed GST-P and alpha-fetoprotein. CONCLUSION: Hepatic progenitor cells have malignant potential in the diethylnitrosamine model of hepatocarcinogenesis, and maintain the immature characteristics of embryonic hepatic stem cells, and still express glutathione-S-transferase and alpha-fetoprotein.