Netrin-1抑制受体Unc5H4诱导人神经母细胞瘤细胞凋亡

Netrin-1 Prevents Apoptosis on SH-SH5Y Cells Induced by Dependence Receptor Unc5H4

目的研究依赖性受体Unc5H4诱导人神经母细胞瘤(neuroblastoma,NB)细胞系SH-SY5Y细胞凋亡而其配体Netrin-1抑制其凋亡诱导的作用。方法人NB细胞系SH-SY5Y细胞中转染Unc5H4质粒,在有/无配体Netrin-1作用下进行平板克隆形成实验,探讨Unc5H4是否诱导细胞凋亡以及诱导细胞凋亡作用是否受Netrin-1蛋白作用的影响。进一步在SH-SY5Y细胞中表达Unc5H4,在阿霉素(ADR)刺激下,在有/无配体Netrin-1蛋白作用时检测Unc5H4蛋白条带的变化,即Unc5H4释放死亡结构域后的截断蛋白诱导细胞凋亡的变化。结果 Caspase-3活性检测结果显示,过表达Unc5H4可诱导SH-SY5Y细胞发生凋亡,其相对Caspase-3活性(180%±20%)明显高于对照组(100%),差异有显著统计学意义(P?0.01)。而加入Netrin-1组细胞的相对Caspase-3活性(112%±14%)明显低于无Netrin-1组(180%?20%),差异有统计学意义(P?0.01),与对照组(100%)相比差异无统计学意义。SH-SY5Y细胞中转染Unc5H4质粒,在同时受到ADR诱导的DNA损伤时,在无Netrin-1蛋白作用下,可以检测到Unc5H4截断蛋白(酶切死亡结构域,60 k Da)的表达,而Netrin-1能够阻止Unc5H4蛋白的截断,从而阻止其诱导凋亡的作用。结论依赖性受体Unc5H4能够诱导人NB细胞凋亡,其配体Netrin-1能够通过阻止其发生蛋白截断而抑制其诱导凋亡的功能,从而使NB细胞获得增殖。

OBJECTIVE To study on the induction of apoptosis in SH-SH5 Y cells by the dependence receptor Unc5H4, while addition of its ligand Netrin-1 can block this effect. METHODS SH-SY5 Y cells were transiently transfected with either a mock plasmid or Unc5H4-expressing construct in the presence or absence of Netrin-1, and then colony formation assay was performed. Whether Unc5H4 induced cell apoptosis or this apoptosis was blocked by Netrin-1 was detected. Further on SH-SY5 Y cells, under stress of adriamycin（ADR） with or without purified Netrin-1 protein, Unc5H4 protein was detected by Western-blot for cleavaged 60 k Da protein. RESULTS Caspase-3 activity assay showed over-expression of Unc5H4 could induce apoptosis in SH-SY5 Y cells, its relative Caspase-3 activity（180±20%） was obviously higher than that of control（100%）（P〈0.01）. While additon of Netrin-1, the relative Caspase-3 activity（112±14%） was much lower than that of group without Netrin-1（180±20%, P？0.01）. The relative caspase-3 activity in Netrin-1 group and control group were no significance. Next, Unc5H4 plasmid was transit transfected on SH-SY5 Y cells. ADR was added for DNA damage. The 60 k Da protein was detected by Unc5H4 antibody, while addition of Netrin-1 protein could block the above. CONCLUSION Dependence receptor Unc5H4 could induce apoptosis in NB cells, while its ligand Netrin-1 could block apotosis by prevention of the cleavage of Unc5H4 to promote cell proliferation.