人羊膜上皮细胞在大鼠受损肝组织中分化为肝细胞

Differentiation of human amnion epithelial cells into hepatocytes in rat injured liver

目的探讨人羊膜上皮细胞在大鼠损伤肝原位植活及向肝细胞分化。方法用胰蛋白酶消化法从羊膜组织中分离人羊膜上皮细胞(hAECs),用流式细胞术和免疫荧光染色进行表型分析和细胞鉴定。腹腔注射D-氨基半乳糖溶液建立大鼠肝损伤模型,随机分为hAECs移植组和对照组,每组20只。造模后24 h用微量注射器于肝左、中、右叶3点分别移植L-DMEM悬浮的hAECs悬液50μL(约1×106个细胞),对照组注射等量L-DMEM。于移植后48 h、1、2和4周处死各实验组5只大鼠,取肝脏制备冰冻切片,用免疫荧光双染色检查hAECs在受损肝原位的植活、分布及其向肝细胞分化的标志物表达。结果 1)FCM分析和免疫荧光染色结果显示,所分离的hAECs表达CD29、CD166及CK19,几乎不表达CD44、CD80、CD86、HLA-DR及波形蛋白;2)免疫荧光双染色结果显示,hAECs移植后1周主要定植于肝小叶且表达AFP,至2周表达CK18,至4周表达Alb。结论 hAECs在大鼠受损肝组织中能被植活且可分化为肝细胞,提示hAECs移植在临床肝病的治疗方面可能具有潜在应用价值。

Objective To investigate the survival and differentiation into hepatocytes of hAECs in rat. Methods hAECs were isolated from human amnion treated with trypsin, and the phenotype and characteristics of immunocyto- chemistry were analyzed by FCM and immunofluorescence staining. Healthy and clean grade female SD rats were ad- ministered intraperitoneal injection of D-galactosamine diluted in normal saline at a dose of 400 mg/kg body weight, to establish liver injury model. Rats were then devided into hAECs group and control group stochastically, with twen- ty in each group. Twenty-four hours after modeling, 50μL cell suspension （approximately 1 ×106 cells suspended in L-DMEM） of hAECs was injected slowly into the left, middle and right lobe respectively with micro-syringe, while equivalent volume of L - DMEM injected into the control group. Results 1 ） Freshly isolated hAECs expressed CD29and CD166; immunofluoresce staining showed that cytokeratin 19 was positive in hAECs, while vimentin was nega- tive. 2）hAECs transplanted into injured liver were located in hepatic lobules at 48 h, and expressed AFP at 1 w, CK18 at 2w, and Alb at 4w after transplantation. Conclusions hAECs xenografted to rat injured liver can differen- tiate into hepatocytes suggesting hAECs may have the potential value for treating clinical liver injury.