突变型 TDP-43（A315T）蛋白诱导S H-SY5Y 细胞凋亡的机制研究

摘要

目的：探讨TDP‐43（A315T ）突变蛋白诱导SH‐SY5Y细胞凋亡的机制。方法采用真核细胞转染技术将Flag‐TDP‐43（w t）及Flag‐TDP‐43（A315T ）质粒转入SH‐SY5Y细胞，通过Western blot检测 TDP‐43截短型表达及自噬水平的变化；PI／Annexin‐V‐FITC检测细胞凋亡率；单丹磺酰尸胺（monodansylcadaverin ，MDC）染色检测细胞自噬空泡的变化。结果与转染Flag‐TDP‐43（wt）组相比，过表达Flag‐TDP‐43（A315T）细胞截短型片段Flag‐TDP‐35及Flag‐TDP‐25表达明显上调，下调细胞内源性Beclin 1水平及LC3Ⅱ／LC3Ⅰ的比值降低。与转染Flag‐TDP‐43（wt）组相比，过表达Flag‐TDP‐43（A315T）诱导SH‐SY5Y细胞自噬性小泡聚集明显减少。与转染Flag‐TDP‐43（wt）组相比，过表达Flag‐TDP‐43（A315T）后SH‐SY5Y细胞凋亡率增加。结论 TDP‐43（A315T ）突变蛋白可通过增加其截短型表达及抑制细胞巨自噬水平诱导SH‐SY5Y细胞凋亡。%Objective To investigate the mechanism of the TDP‐43 (A315T ) regulate autophagy and induced apoptosis mechanism in SH‐SY5Y cells. Methods The recombinant plasmid Flag‐TDP‐43(wt) and Flag‐TDP‐43(A315T) were trans‐fected into SH‐SY5Y cells transfection technique. The cells were collected 24h later ,and the cell total protein were extracted to detect Beclin 1 ,LC3Ⅱ and LC3Ⅰprotein 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 control group transfected Flag‐TDP‐43(wt) ,in the group of overexpression Flag‐TDP‐43(A315T) ,the level of Beclin 1 protein expression was decreased ,and also to the level of LC3Ⅱ /LC3Ⅰ. The percentage of apoptotic cells increased in the Flag‐TDP‐43(A315T) group. Conclusion TDP‐43 (A315T) induced apoptosis of SH‐SY5Y cells by up‐regulation autophagy.