CAJ | 학술논문

目的建立有效的体外诱导人胚胎干细胞(hESCs)分化为肝细胞样细胞的培养体系.方法将H9 hESC细胞株接种到基底膜提取物包被的培养板上,序贯加入含有下列诱导因子的分化培养基诱导分化:100μg/L细胞因子活化素A( activin A)诱导3d,20 μg/L骨形成蛋白4(BMP-4)和10 μg/L碱性成纤维细胞生长因子(bFGF)诱导4d,10μg/L肝细胞生长因子(HGF)诱导5d,最后以含10 μg/L制瘤素M(OSM)及1×10-7 mol/L地塞米松(Dex)的培养液继续诱导4d.于细胞诱导分化第16天,采用逆转录-聚合酶链反应(RT-PCR)及免疫荧光检测分化细胞肝脏特异性基因和蛋白表达水平；过碘酸-雪夫反应(PAS)试验和吲哚氰绿(ICG)摄取试验检测分化细胞是否具备肝细胞功能.结果 activin A、BMP-4、bFGF、HGF、OSM和Dex联合诱导的分化细胞具有类似肝细胞的形态结构,呈多角形或卵圆形；RT-PCR结果显示分化第16天的细胞表达甲胎蛋白(AFP)、细胞角蛋白7(CK7)、细胞角蛋白19(CK19)、肝细胞核因子-4α(HNF4-α)、1-抗胰蛋白酶(AAT)等肝脏特异性基因；免疫荧光化学检测结果显示分化第16天的细胞表达AFP、ALB、CK19、CYP7A1等肝脏特异性蛋白；PAS试验及ICG试验显示分化细胞具备糖原合成和ICG摄取释放等肝细胞功能.结论体外联合多种诱导因子可诱导hESCs定向分化为肝细胞样细胞.
목적 건립유효적체외유도인배태간세포(hESCs)분화위간세포양세포적배양체계.방법 장H9 hESC세포주접충도기저막제취물포피적배양판상,서관가입함유하렬유도인자적분화배양기유도분화:100μg/L세포인자활화소A( activin A)유도3d,20 μg/L골형성단백4(BMP-4)화10 μg/L감성성섬유세포생장인자(bFGF)유도4d,10μg/L간세포생장인자(HGF)유도5d,최후이함10 μg/L제류소M(OSM)급1×10-7 mol/L지새미송(Dex)적배양액계속유도4d.우세포유도분화제16천,채용역전록-취합매련반응(RT-PCR)급면역형광검측분화세포간장특이성기인화단백표체수평；과전산-설부반응(PAS)시험화신타청록(ICG)섭취시험검측분화세포시부구비간세포공능.결과 activin A、BMP-4、bFGF、HGF、OSM화Dex연합유도적분화세포구유유사간세포적형태결구,정다각형혹란원형；RT-PCR결과현시분화제16천적세포표체갑태단백(AFP)、세포각단백7(CK7)、세포각단백19(CK19)、간세포핵인자-4α(HNF4-α)、1-항이단백매(AAT)등간장특이성기인；면역형광화학검측결과현시분화제16천적세포표체AFP、ALB、CK19、CYP7A1등간장특이성단백；PAS시험급ICG시험현시분화세포구비당원합성화ICG섭취석방등간세포공능.결론 체외연합다충유도인자가유도hESCs정향분화위간세포양세포.
Objective To establish an efficient culture system of inducing human embryonic stem cells (hESCs) into hepatocyte-like cells.Methods For hepatic differentiation,hESCs were cultured in basement membrane extract coated plates in culture system supplemented with 100 μg/L activin A for 3 days.The differentiated cells were then cultured in medium containing 20 μg/L bone morphogenetic protein-4 (BMP-4) and 10 μg/L basic fibroblast growth factor (bFGF) for 4 days,and were further differentiated in medium containing 10 μg/L hepatocyte growth factor (HGF) for 5 days.Finally,cells were matured in medium containing 10 μg/L oncostatin M (OSM) and 1 × 10-7 mol/L Dex for another 4 days.At the sixteenth day of cell differentiation,reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence examinations were carried out to detect the hepatic-specific genes and proteins expression of differentiated cells.Periodic acid schiff (PAS) and Indocyanine green (ICG) tests were performed to determine the hepatic-specific functions of differentiated cells.Results During the induction of hESCs,the induced cells showed a significant hepatocyte-like morphology.RT-PCR results showed positive gene expression of alpha fetal protein (AFP),cytokeratin 7 (CK7),cytokeratin 19 (CK19),hepatocyte nuclear factor 4α (HNF4α) and AAT.Immunofluorescence results showed hepatic-specific markers such as ALB,AFP,CK19 and CYP7A1 were also positively expressed.PAS reaction and cellular uptake of ICG indicated the differentiated hESC possessed the hepatic functions of glycogen synthesis and indocyanine grenn uptake.Conclusion The combination of different inducing factors such as activin A,BMP-4,bFGF,HGF,OSM and Dex could successfully induce hESCs to differentiate into functional hepatocyte-like cells.