人羊膜上皮细胞横向分化及脾内移植的初步研究

PRELIMINARY STUDY ON TRANSDIFFERENTIATION OF HUMAN AMNIOTIC EPITHELIAL CELLS AND ITS INTRASPLENIC TRANSPLANTATION

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。

Objective The human amniotic epithelial cells（hAECs） are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes.The hAECs may be a candidate seed cell for liver regeneration.To research the survival and migration in vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene（AAV-GFP） transfection,and to explore the expression of hepatocyte-like function.Methods Thirty nude mice（aging 6-8 weeks,half males and females,and weighing 20-22 g） were randomly divided into 3 groups（groups A,B,and C,n=10）.The mice of groups A and C were made the 2/3 partial hepatectomy model,and the mice of group B underwent open abdominal operation without hepatectomy.The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 10^6/mL and a volume of 0.2 mL;the same volume of normal saline was injected in group C.At 4 hours,the nude mice were sacrificed and the samples of liver,spleen,heart,lung,brain,and kidney were harvested and the general observation,histological observation,and immunofluorescence detection were performed for the hAECs survival,migration,and the functional properties of hepatocytes.Results No tumor tissue was found in liver and spleen of 3 groups,and HE staining showed no tumor cells.There were a lot of roundlike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the liver of group A;no abnormal cells were found in liver and spleen of groups B and C and in kidney,heart,bung,and brain of groups A,B,and C.The GFP＋ cells were detected in the spleen and liver of group A with expressing human albumin,but no GFP＋ cells was found in liver and spleen of groups B and C and in heart,kidney,lung,and brain of groups A,B,and C.Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-like function.It will be beneficial to further identify their biological characteristics.