突变型Notch 3(R90C)蛋白调控自噬诱导VSMC细胞凋亡的分子机制

Mutant Notch 3(R90C) induced VSMCs apoptosis by regnlating autophagy

目的探讨Notch 3(R90C)突变蛋白诱导VSMC细胞凋亡机制。方法采用真核细胞转染技术将Flag-Notch 3(WT)及Flag-Notch 3(R90C)质粒转入大鼠主动脉血管平滑肌细胞A10,通过Western blot检测Beclin 1及LC3Ⅱ/LC3Ⅰ;单丹磺酰尸胺(monodansylcadaverin,MDC)染色检测细胞自噬空泡的变化;PI/Annexin-V-FITC检测细胞凋亡率。结果与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)下调细胞内源性Beclin 1蛋白的表达,LC3Ⅱ/LC3Ⅰ的比值降低。与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)诱导A10细胞自噬空泡聚集减少。与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)使A10细胞凋亡率增加。结论 Notch 3(R90C)突变蛋白可通过抑制细胞巨自噬水平诱导血管平滑肌细胞凋亡。

Objective To investigate the mechanism of Notch3（R90C） induced apoptosis in A10 cells. Methods The re- combinant plasmid Flag-Notch3 （wt） and Flag-Notch3 （R90C） were transfected into A10 cells by eukaryotic cell transfection technique. The cells were collected 24 hours later, and the cells total protein were extracted to detect Beclin 1, LC3 II and LC3 I protein expressions by Western blot. The autophagic vacuoles in the cells were stained with MDC, the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. Results Compared with the transfected Flag- Notch3（wt） group, the level of Beclin I protein expression was decreased in the over-expression Flag-Notch3（R90C） group, and also to the level of LC3 Ⅱ/LC3 I . The percentage of apoptotic cells increased in the Flag-Notch3 （R90C） group. Conclusion Notch3 （R90C） can induce the apoptosis of A10 cells by up-regulation autophagy.