大鼠纤维化肝组织匀浆上清液诱导人脐带间充质干细胞向肝细胞分化的实验研究  

DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS

作　　者：闫成[1,2] 薛改[2] 吴丽颖[2] 刘建芳[2] 侯艳宁[2] 

机构地区：[1]解放军医学院,北京100853 [2]解放军白求恩国际和平医院药剂科

出　　处：《中国修复重建外科杂志》2015年第7期878-883,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：军队"十二五"重点课题资助项目(BWS11J002)~~

摘　　要：目的探讨大鼠纤维化肝组织匀浆上清液对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,HUCMSCs）向肝细胞分化的诱导作用。方法取健康SD大鼠（体重180~220 g）,采用腹腔注射3%硫代乙酰胺生理盐水溶液（200 mg/kg,2次/周,共4周）方法制备大鼠肝纤维化模型,取造模成功的纤维化肝组织用于制备肝纤维化诱导培养基。取第3代HUCMSCs进行分组培养7 d,对照组以含10%FBS的DMEM/F12培养基培养,实验组以含10%FBS及50 g/L肝纤维化诱导培养基上清的DMEM/F12培养基培养。倒置显微镜下观察两组细胞形态学变化;取培养7 d的对照组及实验组细胞,采用Western blot法检测甲胎蛋白（alpha fetoprotein,AFP）、细胞角蛋白18（cytokeratin 18,CK18）、CYP3A4表达情况,采用糖原过碘酸-雪夫（periodic acid-schiff,PAS）染色检测细胞储存糖原的能力,分别采用二乙酰肟比色法及ELISA法测定两组细胞培养液中尿素（blood urea nitrogen,BUN）和白蛋白（albumin,ALB）的含量。结果与对照组长梭形细胞相比,实验组细胞于诱导1 d时即可见到细胞两极回缩,胞体变短;诱导7 d时,细胞呈不规则形或类圆形,胞体丰满。Western blot检测示,对照组细胞未见AFP、CK18、CYP3A4表达;实验组细胞可表达肝细胞特异性标志物AFP、CK18及与代谢功能相关的重要蛋白CYP3A4。对照组上清液中BUN、ALB浓度分别为（0.43±0.07）mmol/L和（8.08±0.41）μg/mL,实验组分别为（2.52±0.20）mmol/L和（41.48±4.11）μg/mL,比较差异均有统计学意义（t=24.160,P=0.000;t=19.810,P=0.000）。PAS染色示,对照组HUCMSCs细胞核呈深蓝色,细胞质呈淡紫色;实验组细胞整个胞体被染成深紫色,细胞核可见。结论大鼠纤维化肝组织匀浆上清液可快速诱导HUCMSCs向肝细胞分化,形成的肝样细胞不仅具备肝细胞表面标志物,同时有肝药酶的表达,并具有储存糖原、合成BUN及分泌ALB的能力,可部分替代肝细Objective To investigate the differentiation potential of human umbilical cord mesenchymal stem cells（HUCMSCs） into hepatocytes induced by rat fibrotic liver tissue extracts. Methods Liver fibrosis was induced in the Sprague Dawley rats（weighting, 180-220 g） by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks; fibrotic liver tissues were used to prepare liver homogenate supernatants.The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum（FBS）（control group） and in DMEM/F12 with 10%FBS and 50 g/L liver homogenate supernatants（experimental group） for 7 days. The morphological changes of the cells were recorded; the protein levels of cytokeratin 18（CK18）, alpha fetoprotein（AFP）, and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff（PAS）staining. Furthermore, the synthesis of albumin（ALB） and blood urea nitrogen（BUN） was measured. Results In experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group.The concentrations of BUN and ALB were（0.43±0.07） mmol/L and（8.08±0.41） μg/mL in the control group and were（2.52±0.20） mmol/L and（41.48±4.11） μg/mL in the experimental group, showing significant differences（t=24.160,P=0.000; t=19.810, P=0.000）. PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. Conclusion HUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers（AFP, CK18, and CYP3A4） and hepatocyte-specific functions of glyc

关 键 词：肝纤维化 人脐带间充质干细胞 细胞分化 微环境 大鼠 

分 类 号：R575.2[医药卫生—消化系统]