体外诱导人脐带间充质干细胞定向分化为肝细胞的研究

Differentiation of human umbilical cord mesenchymal stem cells into functional hepatocyte induced by liver homogenate supernatants in vitro

目的对肝组织勻浆上清液（LHS）体外诱导人脐带间充质干细胞（hUCMSC）定向分化为肝细胞的功能特性进行检测.方法选取第3代hUCMSC,采用LHS作为诱导培养基,实验分为对照组和LHS处理诱导组,每组有3、5、7d三个时间点;诱导后对细胞进行AFP、CK18、TPH2、CYP3A4、Alb、HGF的免疫荧光细胞化学染色;并测定诱导前后CYP3A酶活性、Alb、HGF和尿素的分泌量.结果LHS诱导3d后细胞表达肝特异性蛋白AFP、CK18、TPH2、CYP3A4、Alb和HGF;对照组hUCMSC不同时间点CYP3A酶特异性代谢底物咪达唑仑的代谢量为0.026-0.030ng/mg,LHS诱导3、5和7d后细胞代谢量分别为（30.14±1.19）、（50.20±6.24）和（120.85±15.52）ng/mg,与对照组相比显著升高（P〈0.01）;对照组细胞3、5和7dHGF的分泌量分别为（0.24±0.14）、（0.42±0.20）和（0.18±0.15）ng/mg,LHS诱导后细胞HGF的分泌量分别为（4.06±1.35）、（3.35±0.17）和（3.83±0.42）ng/mg,与对照组相比显著升高（P〈0.01）;LHS诱导后各组细胞尿素分泌量增加14倍以上,与对照组相比显著升高（P〈0.01）;对照组3、5和7dAlb的分泌量分别为（22.66±2.99）、（16.99±2.90）和（15.37±1.86）ng/mg,诱导3、5和7d后Alb的分泌量分别为（36.40±3.53）、（46.66±2.50）和（54.82±4.28）ng/mg,与对照组相比显著升高（P〈0.05或P〈0.01）.结论在体外经LHS诱导后,hUCMSC能够分化为具有成熟肝细胞功能的肝细胞.

Objective To investigate functional characteristics of hepatocytes that differentiating from human umbilical cord mesenchymal stem cells （hUCMSCs） induced by liver homogenate supernatants （LHS）. Methods The 3rd Passage of hUCMSCs were selected and divided into control group and LHS group. At three time points （3d, 5d, 7d） after being induced, the immunofluorescence-cytochemistry staining of alpha-fetoprotein （AFP） , cytokeratin 18 （CK18）, tryptophan hydroxylase 2 （ TPH2 ） ,cytochrome P450 3A4 （CYP3A4 ） ,albumin （ Alb） and hepatocyte growth factor （HGF） were performed, and the CYP3A enzyme activity and levels of HGF, Alb and BUN were detected. Results The cells had expressed liver specific protein at the third day after LHS inducing, including AFP, CK18, TPH2, CYP3A4, Alb and HGF. In LHS group,the metabolism yield of midazolam, the specific metabolic substrates of enzyme CYP3A was increased significantly than that in control group （30. 14 ± 1. 19, 50. 20 ± 6. 24 and 120. 85 ± 15. 52 ng/mg versus 0. 026 to 0. 030 ng/mg, P 〈 0. 01）. Compared with control group, the secretion levels of HGF and Alb in LHS group were increased significantly at 3 time points. Additionally, the urea production of cells in LHS induction group increased 14 times more than that in control group （P〈0. 01）. Conclusion The study has demonstrated that hUCMSCs could differentiate into hepatocytes with mature functions in vitro after LHS induction.