过表达转录因子E2F1对小脑颗粒神经元凋亡的影响

目的探讨过表达转录因子E2F1对小脑颗粒神经元凋亡的影响。方法构建pE2F1表达质粒,转染293细胞验证其表达,双报道基因系统检测其转录活性。体外培养小脑颗粒神经元,钙磷法转染pE2F1表达,转染pcDNA空载体组作为对照,于转染12、24、48h后运用Hoechst 33258染色检测转染细胞的核固缩率（即凋亡率）;转染pE2F1和pcDNA 48h后的小脑颗粒神经元进行高钾和低钾处理12h,统计细胞凋亡率。结果成功构建pE2F1表达质粒,且证实该质粒具有转录活性。与转染pcDNA空载体组比较,转染pE2F1后不同时间点小脑颗粒神经元的凋亡率差异无统计学意义（P〉0．05）。pE2F1转染组和pcDNA转染组高钾处理后的凋亡率分别为（12±4）％和（11±3）％,差异无统计学意义（P〉0．05）;低钾处理后的凋亡率分别为（42±3）％和（30±2）％,差异有统计学意义（P〈0．05）。结论过表达E2F1对小脑颗粒神经元的凋亡无明显影响,但抑制低钾处理诱导的小脑颗粒神经元凋亡。

Effect of E2F1 overexpression on apoptosis of cerebellar granule neurons

Objective To investigate the effect of E2F1 overexpression on the apoptosis of cerebellar granule neurons（CGNs）. Methods The plasmid pE2F1 was constructed and expressed in 293 cells. The transcriptional activity was assessed by 6 × E2F-luciferase reporter gene. The CGNs were cultured in vitro and transfected with pE2F1 and pEGFP using calcium-phosphorylation methods. The CGNs were stained with Hoechst 33258 for apoptosis analysis at 12, 24, 48 h after transfection. Forty-eight hours after transfeetion with pE2F1, CGNs were subjected to 25 or 5 mmol/L KCl treatment for 12 h and then stained with Hoechst 33258 for apoptosis analysis. Results The pE2F1 was successfully constructed and showed a high level of transcriptional activity. Compared with CGNs transfected with pcDNA at different time points, the apoptotic rate of CGNs transfected with pE2F1 had no significant difference（P 〉0. 05 ）. The apoptotic rate of CGNs transfccted with pcDNA or pE2F1 in the condition of 25 mmol/L KCl was （ 12 ± 4 ） % , （ 11 ± 3 ） % respectively, and they had no significant difference. But in the condition of 5 mmol/L KCl, the apoptotic rate of CGNs transfected with pcDNA[ （42 ± 3）% ] was significantly higher than that of CGNs transfected with pE2F1 [ （30 ± 2）% ] （P 〈 0. 05 ）. Conclusion Overexpression of E2F1 had no influence on CGNs, but inhibits apoptosis of CGNs induced by 5 mmol/L KCl treatment.