肝纤维化大鼠血液微环境对人脐带MSCs向肝细胞分化的影响及机制研究  

EFFECT OF BLOOD MICROENVIRONMENT OF RATS WITH HEPATIC FIBROSIS ON DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES AND ITS MECHANISMS

作　　者：闫成 薛改[2] 张伟[2] 韩晓磊[2] 刘建芳[2] 侯艳宁 

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》2016年第6期754-760,共7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：目的探讨肝纤维化大鼠血液微环境对人脐带MSCs(human umbilical cord MSCs,HUCMSCs)向肝细胞分化的影响及作用机制。方法取成年清洁级雄性SD大鼠18只,体质量(200±20)g;其中12只采用腹腔注射硫代乙酰胺方法制备肝纤维化模型,组织学观察确定模型制备成功后取大鼠全血分离血清;剩余6只正常大鼠同法分离血清。ELISA法检测血清中EGF、b FGF、肝细胞生长因子(hepatocyte growth factor,HGF)、抑瘤素(oncostatin M,OSM)含量。取第3代HUCMSCs分组培养7 d,其中肝纤维化血清组(A组)、正常血清组(B组)分别以含5 m L/L肝纤维化大鼠血清、正常大鼠血清的DMEM/F12培养基(含10%FBS)培养;对照组(C组)以DMEM/F12培养基(含10%FBS)培养。培养期间采用倒置显微镜观察细胞形态变化;7 d后取各组细胞行免疫荧光检测肝细胞表面标志物甲胎蛋白(α-fetoprotein,AFP)、细胞角蛋白18(cytokeratin 18,CK18)表达,Western blot检测肝细胞功能蛋白白蛋白(albumin,ALB)、色氨酸2,3-双加氧酶(tryptophan 2,3-dioxygenase,TPH2)、CYP3A4及MAPK/ERK信号通路蛋白(P-ERK)表达,二乙酰肟法测定细胞上清液中尿素氮(blood urea nitrogen,BUN)含量。结果组织学观察示大鼠肝组织符合纤维化改变,模型制备成功。正常大鼠血清中EGF、OSM含量均高于纤维化大鼠,差异有统计学意义(t=14.989,P=0.000;t=37.172,P=0.000);肝纤维化大鼠血清中HGF含量为(1.03±0.12)ng/m L,正常大鼠血清中未检出;两种血清中均未检出b FGF。诱导培养后,倒置显微镜下观察各组细胞形态均未见明显变化且组间无明显差异。培养7 d时,A组细胞可见AFP、CK18绿色荧光,B、C组染色均为阴性。Western blot检测示A组细胞可表达肝细胞功能蛋白ALB、TPH2及CYP3A4,B、C组均未见上述蛋白表达。各组细胞均可表达P-ERK,其中A组P-ERK蛋白相对表达量显著高于B、C组(P<0.05);B、C组间比较差异无统计学意义(P>0.05)。A组细胞上清液中BUN含量亦显著�Objective To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells（HUCMSCs） into hepatocytes and its mechanisms. Methods Eighteen male adult Sprague Dawley rats [weighing,（200±20） g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor（EGF）, hepatocyte growth factor（HGF）, oncostatin M（OSM）, and basic fibroblastic growth factor（b FGF）. Passage 3 HUCMSCs were divided into 3 groups： cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serumand 5 m L/ L serum from rats with hepatic fibrosis（group A）, in DMEM/F12 containing 10% fetal bovine serum and 5 m L/ L serum from normal rats（group B）, and in DMEM/F12 containing 10% fetal bovine serum（group C）. The morphological changes of the cells were observed. The expressions of α-fetoprotein（AFP） and cytokeratin 18（CK18） were detected by immunofluorescence. The protein levels of albumin（ALB）, tryptophan 2, 3-dioxygenase（TPH2）, and CYP3A4 and MAPK/ERK signal pathway protein（P-ERK） were detected using Western blot. The content of blood urea nitrogen（BUN） was measured by diacetyl monoxime method. Results HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were（21.42±0.32） pg/m L and（17.57±0.31） pg/m L respectively, showing significant difference（t=14.989, P=0.000）; the concentrations of OSM were（129.96±0.65） pg/m L and（98.44±1.32） pg/m L respectively, showing significant difference（t=37.172, P=0.000）; the concentrations of HGF were below the detection limit and（1.03±0.12） ng/ m L

关 键 词：肝纤维化 人脐带MSCs 细胞分化 血清 大鼠 

分 类 号：R575.2[医药卫生—消化系统]