MYCN-siRNA和神经生长因子联合诱导神经母细胞瘤细胞分化凋亡的实验研究

目的:探讨应用MYCN-siRNA和神经生长因子(NGF)联合诱导神经母细胞瘤细胞分化凋亡的分子机制。方法:实验分组包括MYCN-siRNA与NGF联合诱导组、NGF诱导组、siRNA诱导组和空白对照组。观察各实验组神经母细胞瘤细胞的形态学变化及细胞的增殖变化,通过Real-TimePCR检测MYCN-mRNA和NSE的表达、Western Blot检测MYCN基因沉默后MYCN蛋白的表达情况。结果:神经母细胞瘤MYCN基因沉默后,RT-PCR检测IMR-32细胞MYCN-mRNA表达的抑制率达到89%;Western Blot检测IMR-32转染MYCN-siRNA后的第3天MYCN蛋白的表达率达到最低,表达率下降至26.6%。在NGF和MYCN-siRNA的共同作用下,神经母细胞瘤细胞出现了较明显的细胞形态学分化,细胞的增殖能力明显下降,IMR-32细胞NSE的表达明显下降。结论:MYCN-siRNA和NGF联合诱导神经母细胞瘤细胞,可使MYCN-mRNA的表达和NSE的表达均明显下降,并促进神经母细胞瘤细胞分化凋亡。

Experimental study of differentiation and apoptosis of neuroblastoma cells co-induced by MYCN-siRNA and NGF

Objective To explore the molecular mechanism of the apoptosis and differentiation of neuroblastoma cells co-induced by MYCN-siRNA and NGF.Methods Four groups were designed in this study,including MYCN-siRNA and NGF co-inducing group,NGF inducing group,siRNA inducing group and negative control group.The morphologic changes and proliferation of cells were observed by inverted phase contrast microscope and MTT cell proliferation assay respectively.Real time PCR was used to detect the expression of MYCN-mRNA and neuron-specific enolase.Suppression of MYCN was measured by western blot after MYCN gene silencing.Results MYCN-siRNA suppression effect was determined by RT-PCR at the ratio of 89%.The lowest expression of MYCN was observed at 72 h after IMR-32 cells silencing by MYCN-siRNA,with the suppression ratio of 26.6% as determined by western blot.MTT assay results showed the cell proliferation capacity obviously decreased.We also observed the expression of neuron-specific enolase obviously decreased by using RT-PCR.Conclusions MYCN-siRNA and NGF co-induced neuroblastoma cells could suppress the expression of MYCN mRNA and NSE,and promote the apoptosis and differentiation of tumor cells.