CAJ | 학술논문

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。
목적：탐토TDP‐43（A315T ）돌변단백유도SH‐SY5Y세포조망적궤제。방법채용진핵세포전염기술장Flag‐TDP‐43（w t）급Flag‐TDP‐43（A315T ）질립전입SH‐SY5Y세포，통과Western blot검측 TDP‐43절단형표체급자서수평적변화；PI／Annexin‐V‐FITC검측세포조망솔；단단광선시알（monodansylcadaverin ，MDC）염색검측세포자서공포적변화。결과여전염Flag‐TDP‐43（wt）조상비，과표체Flag‐TDP‐43（A315T）세포절단형편단Flag‐TDP‐35급Flag‐TDP‐25표체명현상조，하조세포내원성Beclin 1수평급LC3Ⅱ／LC3Ⅰ적비치강저。여전염Flag‐TDP‐43（wt）조상비，과표체Flag‐TDP‐43（A315T）유도SH‐SY5Y세포자서성소포취집명현감소。여전염Flag‐TDP‐43（wt）조상비，과표체Flag‐TDP‐43（A315T）후SH‐SY5Y세포조망솔증가。결론 TDP‐43（A315T ）돌변단백가통과증가기절단형표체급억제세포거자서수평유도SH‐SY5Y세포조망。
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.