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目的人羊膜上皮细胞(human amniotic epithelial cells,hAECs)具有干细胞特性及分化为成熟肝细胞的潜能,有望成为肝细胞移植理想的种子细胞。研究hAECs移植入肝受损裸小鼠脾脏后的存活、迁移情况,探讨其肝细胞特性的表达。方法取顺产产妇自愿捐赠胎盘分离培养hAECs。30只6～8周龄裸小鼠,雌雄各半,体重20～22 g。将实验动物随机分为A、B、C 3组,每组10只。A、C组制备肝切除(常规切除2/3肝)模型,B组仅开腹但未行肝叶切除;术后A、B组分别自脾下极移植细胞密度为5×106个/mL腺相关病毒(adeno-associated virus,AAV)载体介导绿色荧光蛋白(green?uorescent protein,GFP)基因标记的第3代hAECs 0.2 mL,C组注射等量生理盐水。4周后处死动物,取肝、脾、心、肺、脑、肾行大体观察,冰冻切片行组织学及免疫荧光检测,追踪hAECs存活、迁移及肝细胞特性表达。结果 3组裸小鼠肝及脾组织未见异常癌性结节及肿块,HE染色未见肿瘤细胞。A组肝及脾组织HE染色可见大量类圆形、胞质少、核大而深染的细胞;B、C组肝、脾组织及3组肾、心、肺、脑组织均未见异常细胞。荧光显微镜下A组肝、脾组织中均发现绿色荧光信号的hAECs,且大部分表达白蛋白;B、C组肝、脾组织及3组肾、心、肺、脑组织未见绿色荧光细胞。结论 AAV-GFP转染后的hAECs移植入肝受损裸小鼠体内可存活并保持部分肝细胞特性。
The target human amniotic epithelial cells (hAECs) possess the characteristics of stem cells and their potential to differentiate into mature hepatocytes, and are expected to become the ideal seed cells for hepatocyte transplantation. To study the survival and migration of hAECs transplanted into the spleen of nude mice with impaired liver injury and to explore the expression of hepatocyte characteristics. Methods The hAECs were isolated and cultured by voluntary delivery of placenta in pregnant women. 30 6 ~ 8 weeks old nude mice, male and female, weighing 20 ~ 22 g. The experimental animals were randomly divided into A, B, C 3 groups of 10. Groups A and C were prepared for hepatectomy (routine 2/3 hepatectomy), while group B was only laparotomized without hepatectomy. The density of the implanted cells in the spleens of A and B groups was 5 × 106 / mL 0.2 mL of the third generation hAECs labeled with green fluorescent protein (GFP) was transfected with adeno-associated virus (AAV) vector, and the rats in group C were injected with the same amount of normal saline. After 4 weeks, the animals were sacrificed and the liver, spleen, heart, lung, brain and kidney were sacrificed and histological and immunofluorescent staining was performed to observe the survival, migration and hepatocyte characteristics of hAECs. Results No abnormal cancerous nodules and lumps were found in the liver and spleen of the 3 groups. No tumor cells were observed by HE staining. A group of liver and spleen tissue HE staining shows a large number of round, less cytoplasm, nucleus and deep staining of cells; B, C group liver, spleen and kidney, heart, lung, brain tissue no abnormal cells . Green fluorescence hAECs were found in group A liver and spleen under fluorescence microscope, and most of them expressed albumin. There were no green fluorescent cells in liver, spleen and three groups of kidney, heart, lung and brain in groups B and C. Conclusions The hAECs transfected with AAV-GFP can survive in liver-injured nude mice and maintain some hepatocyte characteristics.