转染突变型Notch3基因对线粒体融合蛋白-2表达及下游信号通路的影响

Mutations in Notch3 gene influence expression of Mfn-2 and regulation of specific signaling pathways

目的探讨Notch3基因突变后对线粒体融合蛋白-2(mfn-2)的表达及其下游信号通路的影响,从而进一步阐明伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病(CADASIL)的病理机制。方法利用脂质体法将Notch3野生型重组表达质粒(pcDNA3.1-Notch3)、突变型重组表达质粒(pcDNA3.1-Notch3-R90C)以及pcDNA3.1空载质粒分别瞬时转染到人主动脉平滑肌细胞中并培养。Real-time PCR及western-blot检测各组Notch3的表达;Real-time PCR检测各组mfn-2、bcl-2、survivin基因mRNA的表达;Western-blot检测各组mfn-2蛋白的表达;AV/PI双标法检测各组细胞凋亡情况。结果与空载质粒组和野生型组比较,转染突变型Notch3基因组中mfn-2表达水平明显增加(P<0.05)、bcl-2和survivin基因表达下调(P<0.05)以及细胞凋亡明显增加(P<0.05)。结论 Notch3基因突变后引起的mfn2表达上调及下游凋亡调控基因表达失衡可能是CADASIL发病的重要病理机制。

Objective Cerebral artosomal dominant arteriopath with subcortical infarcts and leukoencephalopathy（CADASIL） is associated with mutations in the Notch3 gene but the causal mechanisms of the disorder remain unclear.This study explored the effects of Notch3 gene mutations on mitochondrial fusion protein 2,bcl-2 and survivin,which may help to further elucidate the pathophysiological mechanisms of CADASIL.Methods Human aortic vascular smooth muscle cells（HVSMC） were transient transfected with wild-type Notch3,mutation-type Notch3-R90C and empty pcDAN3.1 vector according to the Lipofectamine 2000 method.In order to verify the transfection efficiency,real-time PCR and Western blot were used to detect the expression levels of Notch3.The relative gene expression of survivin and bcl-2 was measured using reverse transcription followed by real-time PCR,and the protein expression level of mfn-2 was also examined by Western blot.Moreover,the rate of apoptosis was detected by fluorescein annexin V-FITC/PI double labeling in the three groups.Results Compared with the other two groups,the expression levels of mfn2 mRNA and protein were significantly higher in the mutation-type group（P0.05）.A significant down-regulation in bcl-2 and survivin mRNA expression levels was also found in the mutation-type group（P0.05）.Moreover,Annexin V/PI double staining assay showed the apoptosis rate significantly increased in the mutation-type group（P0.05）.Conclusions Notch3 gene mutation-mediated up-regulation of mfn2 and down-regulation of bcl-2 and survivin may be an essential pathomechanism of CADASIL.