Shanxi Medical University, Taiyuan, Shanxi 030001, China.
J Inorg Biochem. 2009 Nov;103(11):1514-20. doi: 10.1016/j.jinorgbio.2009.06.010. Epub 2009 Aug 14.
Previous studies have demonstrated robust BAK gene silencing via RNA interference (RNAi). To investigate whether BAK RNAi may serve as a co-therapeutic agent in neural cell death, we herein established a cell degeneration model using a human neuroblastoma cell line (SH-SY5Y) treated by aluminum (Al). Combining cell viability assays and expression analyses by QRT (quantitative real-time)-PCR and immunocytochemistry, we selected and validated the optimal small interfering RNA (siRNA) from three candidate siRNAs for the BAK gene. Our data identified siRNA1 as the most effective siRNA; the optimal concentration of the transfection agent was 10nM and the optimal incubation period was 24h. The transfection and knockdown efficiency was 93% and 58%, respectively, which closely correlated with the BAK protein expression. SH-SY5Y cells with BAK knockdown showed a clear resistance against cell death and Al-induced apoptosis. These results indicate that genetic inactivation of BAK could be an effective strategy in delaying the onset of apoptosis in Al-treated cells, and exemplify the therapeutic potential of RNAi-based methods for the treatment of neural cell degeneration.
先前的研究已经证明了通过 RNA 干扰（RNAi）实现 BAK 基因的有效沉默。为了研究 BAK RNAi 是否可以作为神经细胞死亡的辅助治疗剂，我们在此使用铝（Al）处理的人神经母细胞瘤细胞系（SH-SY5Y）建立了细胞退化模型。通过细胞活力测定和 QRT（定量实时）-PCR 和免疫细胞化学表达分析，我们从三个候选 BAK 基因 siRNA 中选择并验证了最佳的小干扰 RNA（siRNA）。我们的数据确定 siRNA1 是最有效的 siRNA；转染剂的最佳浓度为 10nM，最佳孵育时间为 24 小时。转染和敲低效率分别为 93%和 58%，与 BAK 蛋白表达密切相关。具有 BAK 敲低的 SH-SY5Y 细胞对细胞死亡和 Al 诱导的细胞凋亡表现出明显的抗性。这些结果表明，BAK 的遗传失活可能是延迟 Al 处理细胞中细胞凋亡发生的有效策略，并例证了基于 RNAi 的方法在治疗神经细胞退化方面的治疗潜力。
