SelW基因转录后沉默对小鼠骨骼肌细胞内GSH及GPx影响研究

The Influence of SelW on GSH and GPx after Post-Transcriptional Gene Silencing in Mouse Skeletal Muscle Cell

通过降低骨骼肌细胞内硒蛋白W的表达,考察其对GSH和GPx的影响,针对个别研究结果与生物信息学研究结果矛盾的焦点深入研究。利用RNAi敲除小鼠骨骼肌细胞中SelW基因,经定量PCR和Western blot-ting鉴定后,进行WST、GSH含量和GPx活力检测。结果表明,RNAi后目的基因mRNA和蛋白表达量降低了71.9%和68.8%。WST检测结果显示阳性组比对照组下降了21.5%;GSH、GPx检测结果显示阳性组比对照组分别增加了29.76%和47.58%。结果显示,SelW敲除引发细胞的毒性作用,并导致GSH和GPx代偿性增高,与生物信息学关于SelW具有抗氧化功能的研究结果相互验证。

The expression of Selenoprotein W （SelW） in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione （GSH） and the activity of glutathione peroxidase （GPx）. SelW knock-down was performed by RNA interference （RNAi） in cultured muscle cells and verified by Real-time PCR and Western blotting. In addition, cell viability, GSH content and GPx activity were assayed. The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9% and 68.8% relative to control values. WST assay showed that to compare with blank control, the value of positive group dropped 21.5% ; In GSH and GPx assay, to compare with blank control the positive group increased 29.76% and 47.58% separately. In conclusion, SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity. These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.