Lu T, Yang C, Sun H, Lv J, Zhang F, Dong X J
Clinical Laboratory Center of Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, China.
Affiliated Hospital of Xi'an Medical College, Xi'an, Shanxi Province, China.
Genet Mol Res. 2014 Jan 21;13(1):415-24. doi: 10.4238/2014.January.21.9.
Our research demonstrated the potential for mouse bone marrow mesenchymal stem cells (mBMMSCs) to differentiate into hepatocytes in vitro and in vivo. However, the exact mechanism of this process remains unknown. In this study, we investigated the role of the mitogen-activated protein kinase (MAPK) cell-signaling pathway in the differentiation of mBMMSCs into hepatocytes. mBMMSCs were isolated from femurs and tibias, and hepatic differentiation was induced in Isove's modified Eagle's medium supplemented with 10% fetal bovine serum, containing human growth factor and fibroblast growth factor 4. After seven days of induction, morphological characteristics were examined. For inhibition of signaling molecular activities, the inhibitors p38 (SB203580), ERK1/2 (U0126), and MSK1 (H89) were added to the differentiation medium. Real-time polymerase chain reaction and Western blot analysis were used to evaluate the gene expression profiles and protein expression of several markers, including the early specific markers of hepatocytes (AFP and FOXa2), phosphorylated-p38 (p-p38), phosphorylated-ERK1/2 (p-ERK1/2), and phosphorylated- MSK1 (p-MSK1). Expressions of p-p38, p-ERK1/2, and p-MSK1 were effectively inhibited by their respective inhibitors. Expressions of early specific markers, AFP and FOXa2, in the p38, ERK1/2, and MSK1 inhibitor-treated groups were significantly decreased compared to those of the cytokine-induced control. Notably, the expressions of AFP and FOXa2 in the p38 inhibitor group were more obviously reduced than those in the ERK1/2 inhibitor group. The MAPK signaling pathway, especially p38, is sufficient to drive differentiation of mBMMSCs into hepatocytes. This could increase the efficiency of hepatocyte differentiation, which would benefit clinical applications.
我们的研究证明了小鼠骨髓间充质干细胞（mBMMSCs）在体外和体内分化为肝细胞的潜力。然而，这一过程的确切机制仍不清楚。在本研究中，我们调查了丝裂原活化蛋白激酶（MAPK）细胞信号通路在mBMMSCs向肝细胞分化中的作用。从股骨和胫骨中分离出mBMMSCs，并在添加了10%胎牛血清、含有人类生长因子和成纤维细胞生长因子4的伊索夫改良 Eagle培养基中诱导其肝分化。诱导7天后，检查形态学特征。为了抑制信号分子活性，将抑制剂p38（SB203580）、ERK1/2（U0126）和MSK1（H89）添加到分化培养基中。采用实时聚合酶链反应和蛋白质印迹分析来评估几种标志物的基因表达谱和蛋白质表达，包括肝细胞的早期特异性标志物（甲胎蛋白和叉头框蛋白A2）、磷酸化p38（p-p38）、磷酸化ERK1/2（p-ERK1/2）和磷酸化MSK1（p-MSK1）。p-p38、p-ERK1/2和p-MSK1的表达被各自的抑制剂有效抑制。与细胞因子诱导的对照组相比，p38、ERK1/2和MSK1抑制剂处理组中早期特异性标志物甲胎蛋白和叉头框蛋白A2的表达显著降低。值得注意的是，p38抑制剂组中甲胎蛋白和叉头框蛋白A2的表达比ERK1/2抑制剂组更明显降低。MAPK信号通路，尤其是p38，足以驱动mBMMSCs向肝细胞分化。这可以提高肝细胞分化的效率，从而有利于临床应用。
