人羊膜上皮细胞及间充质细胞在大鼠受损肝组织中分化成人肝细胞

发布时间：2019-06-28 16:17

【摘要】： 目的:重症肝病尚无特效疗法,细胞移植治疗肝损伤疾病是一种值得探索的新途径。羊膜上皮和羊膜间充质细胞具有干细胞特性,在体外适当的诱导条件下均可分化为肝细胞,其体内是否可以作为肝细胞供体治疗肝损伤疾病尚未探明。本研究的目的是寻找人羊膜细胞在大鼠肝损伤原位分化为肝细胞的证据,为其作为细胞供体临床治疗肝损伤疾病提供实验依据。 方法:采用二酶消化法从人羊膜组织中分离hAECs和EAMCs,采用流式细胞术(FCM)和免疫荧光染色进行表型分析和细胞鉴定。采用腹腔注射D-氨基半乳糖,按400mg/kg建立大鼠肝损伤模型,随机分为hAECs移植组、hAMCs移植组和对照组,每组20只。造模后24h用微量注射器于肝左、中、右叶3点分别注射L-DMEM悬浮的hAECs或hAMCs悬液50μl(约1×10~6个细胞),对照组注射等量L-DMEM。于移植后48h、1w、2w、3w或4w处死各实验组3～4大鼠,取移植部位行常规病理学检查和行免疫荧光染色或免疫组化染色以观察肝组织病理变化、hAECs和hAMCs在受损肝原位的植活与分布及其甲胎蛋白(AFP)、CK18、CK19和白蛋白(Alb)的表达情况。 结果:①FCM分析结果显示,所分离的hAECs几乎不表达CD44,而hAMCs中则部分表达CD44:免疫荧光染色显示hAECs只表达CK19,而hAMCs只表达波形蛋白。②与对照组比较,hAECs和hAMCs移植组于移植后48h肝门管区嗜碱性小细胞增多,肝细胞坏死较轻;移植后7d肝小叶结构较为完整。③免疫荧光染色结果显示,hAECs肝损伤原位移植后48h主要分散于肝血窦,移植后1w表达AFP,2w表达CK18,2w和4w表达Alb;免疫组化和免疫荧光染色结果显示,hAMCs移植后48h主要分散于肝小叶,1w表达CK19,2w表达CK18,3w仍可检测到人细胞核抗原和Alb。 结论:hAECs和hAMCs在大鼠受损肝组织中能被植活,且可分化为肝细胞,提示这两种人羊膜细胞在临床治疗重症肝损伤疾病方面可能具有重要的应用价值。
[Abstract]:Objective: there is no special effect therapy for severe liver disease. Cell transplantation is a new way worthy of exploration in the treatment of liver injury. Amniotic epithelial cells and amniotic mesenchymal cells have the characteristics of stem cells, which can differentiate into hepatocytes under appropriate induction conditions in vitro. Whether amniotic epithelial cells can be used as liver cell donors in the treatment of liver injury has not been found out. The purpose of this study was to find out the evidence of in situ differentiation of human amniotic cells into hepatocytes in rats with liver injury, and to provide experimental basis for the clinical treatment of liver injury with human amniotic cells as cell donors. Methods: hAECs and EAMCs, were isolated from human amniotic membrane by two enzyme digestion. Flow cytometry (FCM) and immunofluorescence staining were used for phenotypic analysis and cell identification. The rat model of liver injury was established by intraabdominal injection of D-galactosamine according to 400mg/kg. The rats were randomly divided into three groups: hAECs transplantation group, hAMCs transplantation group and control group with 20 rats in each group. 24 hours after modeling, hAECs or hAMCs suspension suspended by L-DMEM (about 1 脳 10 ~ 6 cells) was injected into the left, middle and right lobe of the liver by microsyringe at 3 o'clock, respectively, while the control group was injected with the same amount of L 鈮，

本文编号：2507441