体外诱导人脐带间充质干细胞定向分化为肝细胞的研究

肝脏 ›› 2016, Vol. 21 ›› Issue (9): 733-737.

体外诱导人脐带间充质干细胞定向分化为肝细胞的研究

薛改, 韩华, 陈泽纱, 吴红海, 刘建芳, 侯艳宁

050082 石家庄白求恩国际和平医院药剂科(薛改,吴红海,刘建芳);河北省人民医院妇产科(韩华);河北省儿童医院药剂科(陈泽纱);河北医科大学研究生学院(侯艳宁)

收稿日期:2016-03-21 出版日期:2016-09-30 发布日期:2020-06-19
通讯作者: 侯艳宁,Email:biph2011@163.com
基金资助:
河北省卫生厅青年科技课题(20150122);军队“十二五”重点课题(BWS11J002)

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

XUE Gai, HAN Hua, CHEN Ze-sha, WU Hong-hai, LIU Jian-fang, HOU Yan-ning

Deparment of Pharmacology, Bethune International Peace Hospital, Shijiazhuang 050082, China

Received:2016-03-21 Online:2016-09-30 Published:2020-06-19
Contact: Hou Yan-ning, Email: biph2011@163.com

摘要/Abstract

摘要： 目的对肝组织匀浆上清液(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、7 d 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3 d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0.026～0.030 ng/mg,LHS诱导3、5和7 d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7 d HGF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7 d Alb的分泌量分别为(22.66±2.99)、(16.99±2.90)和(15.37±1.86) μg/mg,诱导3、5和7 d后Alb的分泌量分别为(36.40±3.53)、(46.66±2.50)和(54.82±4.28) μg/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。

关键词: 人脐带间充质干细胞, 诱导分化, 肝组织匀浆上清液, 肝细胞

Abstract: 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.

Key words: Human umbilical cord mesenchymal stem cells, Inducting and differentiation, Liver homogenate supernatants, Hepatocyte

薛改, 韩华, 陈泽纱, 吴红海, 刘建芳, 侯艳宁. 体外诱导人脐带间充质干细胞定向分化为肝细胞的研究[J]. 肝脏, 2016, 21(9): 733-737.

XUE Gai, HAN Hua, CHEN Ze-sha, WU Hong-hai, LIU Jian-fang, HOU Yan-ning. Differentiation of human umbilical cord mesenchymal stem cells into functional hepatocyte induced by liver homogenate supernatants in vitro[J]. Chinese Hepatolgy, 2016, 21(9): 733-737.

参考文献

[1] Secco M,Zucconi E,Vieira NM,et al. Multipotent stem cells from umbilical cord: cord is richer than blood. Stem Cells,2008,26:146-150.
[2] Han YF,Tao R,Sun TJ,et al. Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods. Cytotechnology,2013,65:819-827.
[3] Lin GL,Hankenson KD. Integration of BMP, Wnt, and Notch signaling pathways in osteoblast differentiation. J Cell Biochem,2011,112:3491-3501.
[4] Buyl K,De Kock J,Najar M,et al. Characterization of hepatic markers in human Wharton’s Jelly-derived mesenchymal stem cells. Toxicol In Vitro,2014, 28:113-119.
[5] Yan YM,Xu WR,Qian H,et al. Mesenchymal stem cells from human umbilical cords ameliorate mouse hepatic injury in vivo. Liver Int,2009,29:356-365.
[6] 陈军,刘玉侠,姜文华,等. 体外诱导人脐带间充质干细胞向肝样细胞的分化.中国组织工程研究与临床康复,2010,14:9257-9261.
[7] 任红英,赵钦军,邢文,等. 脐带间充质干细胞体外分化为有功能的低免疫原性肝细胞样细胞. 中国医学科学院学报,2010,32:190-194.
[8] Zhu ZY,Yan JQ,Han T. Long-term in vitro culture and hepatocytic differentiation of human umbilical cord-derived mesenchymal stem cells. Journal of Clinical Rehabilitative Tissue Engineering Research,2009,13:9792-9796.
[9] Campard D,Lysy PA,Najimi M,et al. Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology, 2008,134:833-848.
[10] 马欣,薛改,刘建芳,等. 肝匀浆上清液诱导人脐带间充质干细胞分化为肝样细胞. 中国组织工程研究,2013,17:7877-7884.
[11] Wang XY,Wu HH,Xue G,et al. Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment. Neural Regen Res,2012,7:1925-1930.
[12] Ren H,Zhao Q,Cheng T,et al. No contribution of umbilical cord mesenchymal stromal cells to capillarization and venularization of hepatic sinusoids accompanied by hepatic differentiation in carbon tetrachloride-induced mouse liver fibrosis. Cytotherapy,2010,12:371-383.
[13] Yan Y,Xu W,Qian H,et al. Mesenchymal stem cells from human umbilical cords ameliorate mouse hepatic injuryin vivo. Liver International,2009,29:356-365.
[14] Zhang Z,Lin H,Shi M,et al. Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients. J Gastroenterol Hepatol,2012,27:112-120.
[15] 林沪,张郑,施明,等. 人脐带间充质干细胞治疗失代偿性肝硬化患者的初步研究. 肝脏,2010,17:79-82.
[16] 于宏,王更银,孙殿兴,等. 脐带间充质干细胞经肝动脉灌注治疗失代偿期肝硬化患者. 肝脏,2011,16:185-190.
[17] Choi KS,Shin JS,Lee JJ,et al. In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochem Biophys Res Commun,2005,330:1299-1305.
[18] 严永敏,李里明,李婷芬,等. 人脐带间充质干细胞体外诱导分化为肝细胞样细胞及ERK1/2 通路活化研究. 临床检验杂志,2012,30:591-598.
[19] 潘兴南,魏开鹏. 肝细胞生长因子的浓度和诱导时间人骨髓间充质干细胞向肝细胞分化的影响. 肝脏,2013,18:382 -385.
[20] Campard D,Lysy PA,Najimi M,et al. Native umbilical cord matrix stem cell express hepatic markers and differentiate into heatocyte-like cells. Gastroenterology,2008,134:833-848.
[21] Purnapatre K,Khattar SK,Saini KS. Cytochrome P450s in the development of target-based anticancer drugs. Cancer Lett,2008,259:1-15.
[22] 王忠琼,李昌平,杜光红,等. 多种细胞因子诱导骨髓间充质干细胞向肝细胞的分化. 中国组织工程研究与临床康复,2008,12:4035-4038.