过表达E2F1保护神经元依赖其转录活性

目的探讨E2F1对小脑颗粒神经元死活的影响及机制。方法构建表达质粒pcDNA-E2F1(pE2F1)及突变体pcDNA-E2F1-m132(pm132),转染293细胞验证表达,转染小脑颗粒神经元用6×E2F-luciferase报道基因检测转录活性。转染神经元24 h或48 h后Hoechst 33258核染色,统计转染pE2FI及pm132神经元(GFP阳性细胞)的核固缩率(即凋亡率);或者转染48 h后进行25K、5K处理12hr,统计凋亡率。结果构建质粒表达E2F1及突变体E2F1-m132成功,E2F1具有转录活性而E2F1-m132没有;表达E2F1及E2F1-m132均未诱导神经元凋亡(P>0.05);表达E2F1抑制5K诱导的神经元凋亡(P<0.05),而E2F1-m132没有抑制效应。结论E2F1抑制神经元凋亡依赖其转录活性。

The protective roles for E2F1 in neurons depends on its transactivity

Objective To explore the effect of overexpression of E2F1 on the death and survival of cerebellar granule neurons and the mechanism.Methods The plasmid pcDNA-E2F1 (pE2F1) and pcDNA-E2Fl-m132(pm132)were constructed and expressed in 293 cells.The transcriptional activities for pcDNA, pE2F1 and pm132 were assessed by dual reporter gene system using 6×E2F-luciferase constructs in cerebellar granule neurons (CGNs).CGNs cultured in vitro were transfected with pE2F1 or pm132 for 24 or 48 hours, then neurons were stained with Hoechst 33258 for apoptosis analysis.For the protective assay of E2F1 or pm132, the neurons 48hr after transfection were subjected to 25K, 5K treatment for 12 hr and then stained with Hoechst 33258 for apoptosis analysis.Results Both the plasmids pE2F1 and pm132 expressed successfully.E2F1 showed a high level of transcriptional activity in neurons but the mutation E2Fl-m132 did not.Expression of E2F1 or m132 did not induced CGN apoptosis(P>0.05).Overexpression of E2F1, but not E2Fl-m132, significantly attenuated CGN apoptosis induced by 5K(P<0.05).Conclusion Overepxression of E2F1 rescued CGNs from apoptosis induced by 5K in a transactivity-dependent manner.