Tang Li-jun, Gao Yi, Zhang Zhi, Li Hao, Shan Yu-qiang
Department of Surgery 1, 476th Fuzhou General Hospital of PLA, Fuzhou 350002, China.
Zhonghua Gan Zang Bing Za Zhi. 2005 Sep;13(9):652-5.
To investigate the possibility of the human bone marrow multipotent adult progenitor cells (hMAPCs) to differentiate into hepatocytes with hepatocyte growth factor (HGF)/ fibroblast growth factor-4 (FGF-4) in vitro.
(1) Obtaining the hMAPCs. Bone marrow was obtained from volunteers and then centrifuged through density gradient centrifugation methods. The collected mononuclear cells were cultured through adheret culture to get mesenchymal stem cells (MSCs). The hMAPCs were obtained through collecting and isolating the MSCs by magnetic activated cell sorting (MACS) through depletion selection by use of CD45 and GlyA microbeads. (2) Differentiation of the hMAPCs with HGF+FGF-4. Group A: HGF (20 ng/ml) + FGF-4 (10 ng/ml) induced hMAPCs; group B (positive control group): L-02 human hepatocytes(cell lines); and group C (negative control group): the undifferentiated hMAPCs. (3) The expressions of albumin (Alb), alpha fetoprotein (AFP), cytokeratin-18 (CK-18), and cytokeratin-19 (CK-19) were detected with immunocytochemistry to identify the characteristics of the differentiated cells at different times and the ratio of the positive cells was determined. (4) ALB, AFP, CK-18, and CK-19 expressions of the differentiated cells were detected by RT-PCR assay to investigate the mRNA transcriptions of characteristic hepatic proteins. (5) Alb expressions of the differentiated cells at different times were detected by Western blot on the 21st and 35th days.
(1) The results of immunocytochemistry. The staining of Alb, CK18 were essentially positive in group A. As an early marker of immature hepatocytes, AFP staining was positive on the 7th day but negative in later differentiating periods in group A. (2) The results of RT-PCR. On the 7th day, the differentiated hMAPCs expressed AFP mRNA but were negative in later differentiating periods. On the contrary, the mRNA of Alb and CK-18 were positive at all times. (3) The results of Western blot assay. Alb protein was positive on the 21st day and 35th day.
Under some definite inducing conditions hMAPCs can differentiate into hepatocyte-like cells. They may serve as a potential cell source for liver engineering.
探讨人骨髓多能成体祖细胞（hMAPCs）在体外经肝细胞生长因子（HGF）/成纤维细胞生长因子-4（FGF-4）诱导分化为肝细胞的可能性。
（1）获取hMAPCs。从志愿者处采集骨髓，采用密度梯度离心法进行离心。收集的单个核细胞通过贴壁培养获得间充质干细胞（MSCs）。利用抗CD45和GlyA微珠通过磁激活细胞分选（MACS）进行去除选择，收集并分离MSCs从而获得hMAPCs。（2）hMAPCs经HGF+FGF-4诱导分化。A组：HGF（20 ng/ml）+FGF-4（10 ng/ml）诱导hMAPCs；B组（阳性对照组）：L-02人肝细胞（细胞系）；C组（阴性对照组）：未分化的hMAPCs。（3）采用免疫细胞化学法检测白蛋白（Alb）、甲胎蛋白（AFP）、细胞角蛋白-18（CK-18）和细胞角蛋白-19（CK-19）的表达，以鉴定不同时间分化细胞的特征并测定阳性细胞比例。（4）采用逆转录-聚合酶链反应（RT-PCR）法检测分化细胞的ALB、AFP、CK-18和CK-19表达，以研究特征性肝蛋白的mRNA转录情况。（5）在第21天和第35天通过蛋白质免疫印迹法检测不同时间分化细胞的Alb表达。
（1）免疫细胞化学结果。A组中Alb、CK18染色基本呈阳性。作为未成熟肝细胞的早期标志物，AFP染色在第7天呈阳性，但在A组后期分化阶段呈阴性。（2）RT-PCR结果。在第7天，分化的hMAPCs表达AFP mRNA，但在后期分化阶段呈阴性。相反，Alb和CK-18的mRNA在所有时间均呈阳性。（3）蛋白质免疫印迹法检测结果。Alb蛋白在第21天和第3天呈阳性。
在一定诱导条件下，hMAPCs可分化为肝细胞样细胞。它们可能成为肝脏工程的潜在细胞来源。
