神经酰胺在辐射诱导的秀丽隐杆线虫生殖细胞旁效应中的机制研究

The role of ceramide in radiation-induced bystander effect on germ cells in Caenorhabditis elegans

目的利用秀丽隐杆线虫为研究对象,以单粒子束为辐射源,从活体水平探究神经酰胺在辐射旁效应中的作用。方法以野生型N2和突变体L4期线虫的后食道球为辐射部位,分别给予2 000个质子的辐照剂量,检测和分析成虫的生殖腺凋亡细胞以及基因表达水平。结果定点辐射N2的后食道球可显著诱导线虫旁区生殖腺细胞凋亡的增加(t=9.007,P<0.05),但在神经酰胺合酶基因突变体中未表现出这一现象(P>0.05)。实时定量PCR结果显示,辐射诱导N2和神经酰胺合酶基因突变体lagr-1(gk327);hyl-1(ok976)线虫中凋亡核心通路基因egl-1和ced-13表达量的上升,但两品系线虫间的差异无统计学意义(P>0.05);辐射可诱导N2和DNA损伤检验点突变体hus-1(op241)中hyl-1和lagr-1表达量的上升,且两品系间的差异无统计学意义(P>0.05)。非受体络氨酸激酶abl-1突变可显著增加辐射诱导的旁区细胞凋亡(t=13.241,P<0.05),而当神经酰胺合酶基因与abl-1同时突变时,凋亡增加的现象被抑制(t=13.462,P<0.05)。结论神经酰胺参与线虫体内辐射引起的旁区凋亡过程,且可能与凋亡核心通路中的促凋亡蛋白egl-1和ced-13以及DNA损伤响应通路中的HUS-1协同发挥作用。抑凋亡基因abl-1与神经酰胺合酶在凋亡过程中表现出拮抗关系。

Objective To investigate the role of ceramide in radiation induced bystander effect (RIBE) in vivo by irradiating Caenorhabditis elegans with proton microbeam. Methods The posterior pharynx of wide-type N2 and genetically mutated L4-staged worms was irradiated with 2 000 particles, then germ cell apoptosis and gene expression were analyzed. Results Point-fixed radiation to posterior pharynx of N2 worms significantly increased the number of apoptotic germ cells (t=9.007, P<0.05), but not in the worms with deletion mutants of ceramide synthase (CerS) genes (hyl-1 and lagr-1)(P>0.05). Real-time quantitative PCR assay indicated that in both N2 and lagr-1(gk327);hyl-1(ok976) worms, the genes of egl-1 and ced-13 in core apoptosis pathway were up-regulated after radiation to posterior pharynx, without statistical significance between these two strains (P>0.05). The gene expression levels of hyl-1 and lagr-1 were increased in both N2 and hus-1(op241) worms after radiation with no statistical significant difference between two strains (P>0.05). Furthermore, radiation-induced germ cell apoptosis was increased in abl-1(ok171) worms (t=13.241, P<0.05), rather than in lagr-1(gk327);hyl-1(ok976);abl-1(ok171) worms (t=13.462, P<0.05). Conclusions Ceramide is required for RIBE on germ cell apoptosis in C. elegans, and it might play function together with egl-1 and ced-13 in core apoptosis pathway and HUS-1 in DNA damage response pathway. But ceramide had antagonistic relationship with anti-apoptotic protein abl-1 in germ cell apoptosis.