HGF及G-CSF诱导大鼠骨髓间充质干细胞分化为肝样细胞

HGF and G-CSF induce the differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells

目的探讨肝细胞生长因子(HGF)以及粒细胞集落刺激因子(G-CSF)可否诱导大鼠骨髓间充质干细胞(BM-MSCs)分化为肝样细胞。方法以贴壁法分离、培养大鼠BM-MSCs,流式细胞术鉴定表面标志。实验分为:对照组(10%FBS),0.1μmol/L G-CSF干预组,20 ng/mL HGF干预组,HGF联合G-CSF共同干预组(0.1μmol/LG-CSF+20 ng/mL HGF),诱导后第7天、14天和21天提取细胞总RNA以及总蛋白后进行RT-PCR和Western blot检测白蛋白(ALB)和甲胎蛋白(AFP)基因mRNA转录和蛋白水平。结果 20 ng/mL HGF干预组和HGF联合G-CSF共同干预组P3代BM-MSCs诱导后细胞形态似肝样细胞。BM-MSCs表面标志物:CD34-PE 0.3%,CD45-FITC0.1%,CD90-FITC 99.6%,CD105-PE 99.8%。20 ng/mL HGF干预组和HGF联合G-CSF共同干预组检测到白蛋白(ALB)和甲胎蛋白(AFP)基因mRNA转录水平及蛋白水平表达。在第7、14、21天,随时间增加ALB表达量递增、而AFP表达量递减,且同一时间两组表达量均有差异(P<0.05)。结论 HGF能诱导大鼠BM-MSCs分化为肝样细胞,G-CSF对HGF诱导大鼠BM-MSCs分化为肝样细胞有协同作用。

Objective To investigate if the hepatocyte growth factor （HGF）and granlocyte-colony stimulating factor （G-CSF） induce differentiation of rat bone marrow mesenchymal stem cells（BM-MSCs） into hepatoeyte-like cells. Methods The BM-MSCs were isolated and cultured by adherent method,then identified by flow cytometry. The BM- MSCs of the third passage were used as four groups-control group （ 10% FBS） and intervation groups （0. 1 μmol/L G-CSF;20 ng/mL HGF;0. 1 μmol/L G-CSF ＋20 ng/mL HGF）. The mRNA and protein were extracted on 7,14,21 days,then detected by RT-PCR and Western blot. Results The third passage BM-MSCs of intervation groups （20 ng/mL HGF and 0. 1 μmol/L G-CSF ＋20 ng/mL HGF） got hepatocyte like feafure after induction. The results tested by flow cytometry analysis were as follows： CD34-PE 0. 3% ,CD45-FITC 0. 1% ,CD90-FITC 99. 6% ,CD105-PE 99. 8%. The mRNA and protein of ALB and AFP were detected respetively by RT-PCR and Western blot on day 7, 14,and 21 from inervation groups （10% FBS ＋20 ng/mL HGF;10% FBS ＋0. 1 μmoL/L G-CSF ＋20 ng/mL HGF）,however,the amount of ALB increased as time went by,while AFP decreased to extremely low on day 21. Meaningful- ly, the difference between the two groups is significant （P 〈 0. 05 ）. Conclusions HGF can induce the differentiation of BM-MSCs into hepatocyte-like ceils, which can be significantly enhanced by G-CSF.