EGFP-LC3稳定细胞系的建立及对Aβ自噬效应的研究

Establishment of EGFP-LC3-HEK293 Stable Cell Line and the Role in Aβ-induced Autophagy

该文建立了稳定表达EGFP-LC3的HEK293细胞系,以10μmol/L的Aβ肽及其不同疏水性氨基酸含量的截短形式处理细胞24 h,计数LC3的荧光斑点数;Western blot检测LC3B表达量的变化;MTT检测特定Aβ诱导细胞自噬后对细胞活性的差异;用透射电镜确认自噬体的细胞超微结构。结果显示,G418(700μg/m L)筛选6周后,建立了稳定表达EGFP-LC3的HEK293细胞系;Aβ25-35、Aβ40和Aβ42诱导细胞内LC3荧光斑点的效果较明显;Western blot结果显示LC3B的酯化,即LC3BI向LC3BII转变;MTT检测发现,与Aβ40相比,Aβ42处理后伴随自噬的细胞毒性更强;电镜可以见到Aβ诱导的自噬小体。提示,Aβ肽及其截短的疏水性片段均可诱导自噬,且诱导自噬的效果与疏水性氨基酸含量无关;同时,Aβ42细胞损伤强于Aβ40。该研究为进一步探讨AD自噬机制提供了实验基础。

In this study, an EGFP-LC3-HEK293 stable cell line has been established. The cells were incubated with 10 μmol/L of Aβ and the truncated peptides with various percentages of hydrophobic amino acids（Aβ40, Aβ42, Aβ1-11, Aβ12-24, Aβ25-35 and Aβ35-42） for 24 h. The expression of LC3 was detected by fluorescence microscopy and Western blot, cell viability was measured via MTT assays and the ultrastructural morphology was studied by transmission electron microscopy（TEM）. As a result, the best concentration of G418 was 700 μg/m L for EGFP-LC3 stable clone screen. The quantitative immunofluorescence microscopy data showed that Aβ25-35, Aβ40 and Aβ42 induced autophagy significantly in EGFP-LC3-HEK293 stable cells. MTT assays showed much stronger cytotoxicity in Aβ42-treated group than in Aβ40 group whereas both peptides induced autophagy. LC3BII/I shifting which indicated LC3 B lipidation was detected via Western blot. Aβ-induced autophagosomes could be observed by TEM. Our data suggests that autophagy can be induced by Aβ peptides and the truncated forms of hydrophobic amino acids fregment, but the autophagy induction is not correlated with the percentage of hydrophobic amino acids. Furthermore, Aβ42 is much more cytotoxic than Aβ40 during autophagy induction. Based on this study, our research lay the foundation for further mechanisms of autophagy in Alzheimer＇s disease.