体外诱导可分解草酸成人骨髓间充质干细胞向肝细胞分化的研究

In vitro hepatic differentiation of oxalate-degrading human bone mesenchymal stem cells

目的体外培养并转染了产甲酸草酸杆菌（Ox-F）草酸分解基因h℃和oxe的成人骨髓间充质干细胞（hMSCs—fre—oxe），并诱导其向肝细胞分化。方法体外培养转染了草酸分解基因的P4代hMSCs，并通过含40μg/L肝细胞生长因子（HGF）＋10μg／L胰岛素样生长因子（FGF）-4所组成的培养液诱导其向肝细胞分化。在分化过程中通过显微镜观察分化细胞形态变化。以实时定量聚合酶链反应（Real—timePCR）、Westernblot技术和免疫细胞化学技术检测细胞内肝细胞特异标志物甲胎蛋白（AFP）、白蛋白（ALB）、细胞角蛋白-18（CK-18）和目的基因frc、OXC及其产物融合蛋白mye—FCOAT、flag—OCOAD的表达。成熟肝细胞L-02和转染了空病毒载体的hMSCs（hMSCs．vector）分别作为阳性对照组和阴性对照组。结果可分解草酸的hMSCs—frc—OXC在体外可成功培养，细胞经上述培养液诱导后，细胞形态逐渐变短，变成多角形。Real—timePCR可检测在诱导组细胞中AFP、ALB、CK—18和草酸分解基因frc、OXe表达，而未经诱导细胞组AFP、ALB、CK-18则未表达，未转染目的基因的细胞组fre、oxe未见表达。免疫细胞化学检测诱导28d后细胞可表达ALB，未经诱导细胞组ALB表达呈阴性。Westernblot技术检测诱导第14、28天细胞中AFP、CK-18表达较未诱导组明显增加（P〈0．05），与成熟肝细胞相似，并表达myc-FCOAT、flag—OCOAD，而未转染目的基因细胞组表达呈阴性。结论含有草酸分解基因的hMSCs—frc—OXC体外通过一定条件诱导可以向肝细胞方向分化，经诱导分化后细胞仍可成功表达frc和oxe基因及其产物。

Objective To induce the differentiation of human bone mesenchmal stem cells with ox- alate-degrading genes （hMSCs-fre-oxe） into hepatoeyte-like cells in vitro. Methods The hMSCs-frc-oxc in passage 4 were cultured and induced to differentiate into hepatocyte-like cells in differentiation medium sup- plemented with 40 μg/L hepatoeyte growth factor （HGF） and 10μg/L fibroblast growth factors-4 （FGF-4）. The morphological changes of cells were observed microscopically. The RNA and protein levels of hepatic markers [ alpha fetal protein （ AFP）, cytokeratin-18 （ CK-18 ） and albumin （ALB） ] and oxalate-degrading genes （frc and oxc） were detected by using immunocytochemistry, Western blotting and real-time quantita- tive polymerase chain reaction （Real-time PCR）. L-02 cells and transfected mock-vehicle hMSCs （hMSCs- vector） were used as positive and negative controls, respectively. Results The hMSCs-frc-oxc were cultured successfully in vitro. After induction, hMSCs-frc-oxc gradually transformed into polygonal cells. Real-time PCR revealed the expression of hepatic markers （AFP, ALB and CK-18） and oxalate-degrading genes （frc and oxc） in hMSCs-frc-oxc after inducting for 14 days and 28 days, whereas hMSCs cultured alone did not show liver specific gene expression. Immunocytochemistry revealed positive ALB expression in cells differen- tiating for 28 days. Western blotting revealed that the expression levels of AFP and CK-18 in differentiated cells were higher than in undifferentiated cells （ P 〈 0. 05 ）. The differentiated hMSCs-frc-oxc also expressed the oxalate-degrading genes products （myc-FCOAT and flag-OCOAD）. Conclusion The hMSCs with ox- alate-degrading genes could be induced to differentiate into hepatocyte-like cell by HGF and FGF-4 at certain concentrations and the hepatic differentiated hMSCs-frc-oxc could express the oxalate-degrading genes and their products.