ATRA诱导神经母细胞瘤细胞分化及TrkA表达的研究

Establishment of neuroblastoma (NB) cell line and induction of cell differentiation with all-trans retinoic acid

目的 观察全反式维甲酸 (all transretinoicacid ,ATRA)体外对神经母细胞瘤 (neurob lastoma ,NB)细胞增殖的抑制和可能的分子机制及NGF TrkA信号传导途径在NB细胞分化中的作用。方法 取本院住院 2岁男性NB患儿新鲜手术标本 ,进行原代细胞培养 ,并对培养细胞进行分离与纯化 ,建立细胞系 ,作为细胞模型。通过台盼蓝拒染法计数活细胞 ;观察ATRA干预前后细胞形态学变化 ;RT PCR分析TrkA表达情况。结果 ATRA作用NB细胞后 ,细胞形态发生显著变化 ,并可抑制细胞增殖 ,2d时TrkA表达增加 ,4d时出现TrkA表达的明显增加。结论 所建立的NB细胞系为可诱导分化型 ,即N型 ;≥ 5 μmol/L ,ATRA对NB细胞体外增殖有抑制作用并诱导其分化且表现剂量依赖关系 ;ATRA可诱导NB细胞分化成熟及TrkAmRNA表达水平增高 ,可能是ATRA体外诱导NB细胞分化逆转的分子生物学机制之一。

Objective The current study was designed to determine the possible molecular mechanisms of ATRA-induced growth inhibition and the effect of NGF-TrkA signal transduction pathway on NB cell differentiation and the possible mechanisms of NB spontaneous regression.Methods NB tumor tissues of inpatients were collected, primary cell culture was performed, and the cells were separated and purified to be cell line as a cell model. Trypan blue exclusion was used to count the alive cells; morphological changes were observed under the phase-contrast microscope.Results By day 2, the alive cells of Group A and B decrease after treatment of ATRA; Morphological changes of NB cells were also observed by phase contrast microscopy. The results of this study also showed that the TrkA expression begin to increase from day 2 and reach its peak at day 8.Conclusion NB cells treated by 5?μmol/L and 20?μmol/L concentration ATRA differentiated to neuron-like cells and the cell growth was inhibited. The NB cell line can be used as differentiation-induction model. ATRA can inhibit cell growth and induce its differentiation in dose-dependent fashion. This may be one of the mechanisms of spontaneous regression of NB.