NF-κB和Ubc9在HGF和β-NGF诱导骨髓基质干细胞分化为肝细胞中的作用

ROLE OF NF-κB AND Ubc9 IN DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELL TO HEPATOCYTE INDUCED BY HGF AND β-NGF

目的探讨核因子κB(NF-κB)和泛蛋白连接酶(Ubc9)在肝细胞生长因子(HGF)和β-神经生长因子(β-NGF)诱导骨髓基质干细胞(BMSCs)向肝样细胞分化中的作用。方法取小鼠股骨骨髓,直接贴壁法分离纯化BMSCs,传代,取第3代BMSCs,分为HGF组(胎肝细胞加入30μg/L的HGF诱导14d)、β-NGF组(胎肝细胞加入50μg/L的β-NGF诱导14d)、阳性对照组(胎肝细胞培养12d)和阴性对照组(不加诱导剂的BMSCs)。显微镜下观察诱导不同时间各组细胞形态变化,应用免疫荧光法鉴定肝细胞特异表达蛋白α1-抗胰蛋白酶(AAT)表达,免疫荧光及Realtime-PCR方法检测诱导14d后细胞NF-κB、Ubc9蛋白和mRNA的表达。结果显微镜下观察显示,经过HGF与β-NGF诱导14d后部分细胞呈圆形或卵圆形,与阳性对照组细胞形态一致;免疫荧光结果显示,经HGF与β-NGF诱导14d后细胞胞质AAT均呈阳性表达,部分细胞胞质与胞核同时呈阳性表达。与阴性对照组比较,经HGF与β-NGF诱导后NF-κB和Ubc9主要在细胞胞质中呈阳性表达,荧光强度随细胞形态向肝细胞分化呈现逐渐增强趋势。Realtime-PCR结果显示,HGF组、β-NGF组NF-κB、Ubc9mRNA的表达均较阳性对照组增高,差异有显著性(F=2 272.6、866.4,P<0.01)。结论 HGF和β-NGF诱导BMSCs向肝细胞分化过程中NF-κB和Ubc9均呈阳性表达,在BMSCs向肝细胞诱导分化过程中发挥重要作用。

Objective To study the role of NF-κB and Ubc9 in the differentiation of BMSCs into hepatocyte-like cells induced by hepatocyte growth factor（HGF）andβ-nerve growth factor（β-NGF）in vitro. Methods Mouse femur bone marrow was taken,and a direct adherent method was used to separate and purify BMSCs and transfer of culture.The third generation of BMSCs was taken and divided into HGF group-fetal liver cells were added to 30μg/L HGF and induced for 14d;β-NGF group-fetal liver cells were added to 50μg/L and induced for 14d;positive-control group-β-NGF fetal liver cell culture for 12days;negativecontrol group-BMSCs without inducing agent.Changes in cell morphology at different time of induction were inspected under a microscope.Using immunofluorescence,hepatocyte specific proteinα1-antitrypsin（AAT）protein expression was identified,and the expressions of NF-κB and cell Ubc9 protein at 14 days after induction were detected using immunofluorescence and Realtime-PCR.Results Fourteen days after HGF andβ-NGF induction,microscopy showed some cells in theβ-NGF group appeared as round or oval in shape,being consistent with the cell appearance in the positive control group,and immunofluorescence demonstrated cytoplasm AAT was positively expressed,and partial cytoplasm and nucelus showed positive expression at the same time.Compared with the negative-control group,positive expressions of NF-κB and Ubc9 were mainly in cytoplasm,the fluorescence intensity showed a gradual strengthening trend with cell morphology differentiates to hepatocyte.Realtime-PCR results showed that the expressions of Ubc9 mRNA and NF-κB in HGF group andβ-NGF group were higher than that in the positive-control group,the differences were significant（F=2 272.6,866.4;P〈0.01）. Conclusion In the process of HGF-andβ-NGF-induced bone marrow stromal stem cells to hepatocyte differentiation,the expressions of NF-κB and Ubc9 are positive,which play an important role in the cell differentiation.