Jiang Ze-Sheng, Gao Yi, Mu Ning
Department of General Surgery, Zhujiang hospital, Southern Medical University, Guangzhou 510282, China.
Zhonghua Yi Xue Za Zhi. 2007 Feb 6;87(6):414-8.
To investigate the possibility of multipotent adult progenitor cells from human bone marrow ZHJ-MAPC to differentiate into hepatocytes by co-culture with human hepatocyte line L02 in vitro.
(1) Multipotent adult progenitor cells from human bone marrow, screened in Zhujiang hospital, named ZHJ-MAPC, were divided into 2 parts. Some ZHJ-MAPCs undergo co-culture with human hepatocytes of the line L-02 in the manner without cell to cell contact. On the days 1, 3, 5, and 7 immunocytochemistry was used to detect the expression of albumin (ALP), alpha-fetoprotein (AFP), cytokeratin (CK)-18, and CK-19, characteristic of hepatocyte. Some ZHJ-MAPC were labeled with Certified Functional Safety Expert (CFSE), a fluorescent reagent, and were mixed with L02 cells so as to undergo co-culture in the manner with cell to cell contact. Then the mixed cells were seeded on specialized dish for detection by Laser Scanning Confocal Microscope (LSCM). 5 days later, the cells were double stained with SABC-Cy3. The expression of ALP, AFP, and CK-18 in the ZHJ-MAPC were observed under LSCM. Separately cultured L02 hepatocytes served as positive control s and separately cultured ZHJ-MAPC served as negative controls.
(1) Results of co-culture without cell to cell contact. On the first day, the ZHJ-MAPC expressed high level of AFP and then the AFP expression tapered daily. The expression of ALP was very weak on day 1 and then increased and peaked on day 5. The expression of CK-18 began to appear on day 5 and peaked on day 7. The expression of CK-19 was always negative. (2) Results of co-culture with cell to cell contact. On day 5 there were three colors of fluorescence under LSCM. Yellow cells represented the ZHJ-MAPC differentiating into hepatocytes; the green cells were undifferentiating ZHJ-MAPC; and the red cells were L02 hepatocytes. The result showed that ALP and CK-18 were expressed in many ZHJ-MAPC; AFP appeared only in a few cells.
ZHJ-MAPC can be induced to differentiate into mature hepatocyte-like cells by co-culture, either with or without cell to cell contact.
探讨人骨髓多能成体祖细胞ZHJ-MAPC与人类肝细胞系L02体外共培养分化为肝细胞的可能性。
（1）在珠江医院筛选的人骨髓多能成体祖细胞，命名为ZHJ-MAPC，分为两部分。部分ZHJ-MAPC与L-02人肝细胞进行非细胞接触方式的共培养。在第1、3、5和7天，采用免疫细胞化学法检测白蛋白（ALP）、甲胎蛋白（AFP）、细胞角蛋白（CK）-18和CK-19的表达，这些是肝细胞的特征性指标。部分ZHJ-MAPC用荧光试剂羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记，与L02细胞混合进行细胞接触方式的共培养。然后将混合细胞接种于专用培养皿，用激光扫描共聚焦显微镜（LSCM）检测。5天后，细胞用SABC-Cy3进行双重染色。在LSCM下观察ZHJ-MAPC中ALP、AFP和CK-18的表达。单独培养的L02肝细胞作为阳性对照，单独培养的ZHJ-MAPC作为阴性对照。
（1）非细胞接触共培养结果。第1天，ZHJ-MAPC AFP表达水平较高，随后AFP表达逐日下降。第1天ALP表达非常弱，随后升高并在第5天达到峰值。CK-18表达在第5天开始出现并在第7天达到峰值。CK-19表达始终为阴性。（2）细胞接触共培养结果。第5天，LSCM下有三种荧光颜色。黄色细胞代表ZHJ-MAPC分化为肝细胞；绿色细胞为未分化的ZHJ-MAPC；红色细胞为L02肝细胞。结果显示，许多ZHJ-MAPC表达ALP和CK-18；AFP仅在少数细胞中出现。
ZHJ-MAPC通过共培养，无论有无细胞接触，均可诱导分化为成熟的肝细胞样细胞。
