Γ干扰素诱导功能性TrkA的表达激活NGF/TrkA传导通路诱导原代神经母细胞瘤细胞分化的实验研究

Interferon-gamma induced activated TrkA expression and caused neuronal differentiation in aggressive primary human neuroblastoma via NGF/TrkA pathway

目的在骨髓转移的神经母细胞瘤(Neuroblastoma,NB)细胞及人KP-N-RT细胞系中分别应用IFN-γ、NGF及联合应用IFN-γ和NGF作为诱导剂诱导NB细胞分化。方法NB骨髓转移细胞的原代培养以及人KP-N-RT细胞系培养10d。第10天在原代培养细胞及KP-N-RT细胞中应用RT-PCR法检测TrkA表达水平;同时应用Western法和免疫沉降法检测KP-N-RT细胞中TrkA及磷酸化TrkA蛋白表达的不同。结果第10天观察细胞形态学变化,在NB原代培养及KP-N-RT细胞中同时负荷IFN-γ和NGF的细胞表现出比单独负荷IFN-γ或NGF更加显著的形态学分化(P<0.01)。前者细胞增殖抑制明显(P<0.01)。在IFN-γ组,TrkAmRNA表达增加,NGF组未见明显改变,联合用药组有明显减少或消失。在KP-N-RT细胞中,在25IU/mLIFN-γ处理的细胞中用NGF刺激5min可致磷酸化TrkA蛋白表达增加。结论IFN-γ通过磷酸化NGF受体激活NGF/TrkA信号传导系统诱导NB细胞的分化,甚至在侵袭性表型的NB中也有相似结果。

[Objective] To induce the differentiation of neuroblastoma cells from metastastic bone marrow and human KP-N-RT cells by interferon-γ（IFN-γ）, nerve growth factor （NGF）, and a combination of IFN-γ and NGF respectively. [Methods] Metastatic Neuroblastoma cells from bone marrow of a primary stage Ⅳ NB patient and human KP-N-RT cell line were cultured for 10 days. On day 10, RT-PCR was performed to test the TrkA levels in primary cultured cells as well as KP-N-RT cells. Western Blot and immunoprecipltation were performed in the lysate treated with IFN-γ and NGF. [Results] The simultaneous loading of NGF with IFN-γ caused more prominent neurite outgrowth than independent treatment with either IFN-γ or NGF in both KP-N-RT cells and primary cultured NB cells （P〈0.01） on day 10. The cell proliferation was inhibited significantly in the former （P〈0.01）. TrkA mRNA level increased in IFN-γ group, there was no observable change in the NGF group and was obvious decreased or dismissed in the combination group. In KP-N-RT cells, NGF stimulation can cause phosphorylated TrkA protein with IFN-γ pretreatment. [Conclusions] IFN-γ can activate the NGF/TrkA signal transduction pathway to induce the differentiation of neuroblastoma cells by phosphorylating the functional NGF receptor even in the aggressive phenotype of Neuroblastoma.