RNA干扰抑制脊髓源星形胶质细胞sIL-6R基因表达的实验研究  

Research on RNA Interference Suppressing Expression of sIL-6R of Spinal Cord-derived Astrocytes

作　　者：林健泽[1] 沈哲[1] 高明勇[1] 余铮[1] 郭伟壮[1] 李振宇[1] 陈扬[1] 

机构地区：[1]深圳市第二人民医院脊柱外科,广东深圳518035

出　　处：《深圳中西医结合杂志》2012年第1期8-12,F0003,共6页Shenzhen Journal of Integrated Traditional Chinese and Western Medicine

基　　金：深圳市科技计划项目(200903049)

摘　　要：目的:靶向白细胞介素6受体(sIL-6R)基因构建具有高干扰效率的RNA干扰载体,观察其对脊髓源星形胶质细胞sIL-6R基因表达抑制效果。方法:靶向sIL-6R基因设计3条短发夹样RNA(short hairpin RNA,shRNA)的寡核苷酸片段,构建sIL-6R siRNA重组质粒真核表达载体,转染脊髓源星形胶质细胞,筛选出对sIL-6R基因干扰效率最高的sIL-6R siRNA,用免疫组化染色、RT-PCR及Western blot技术观察其对脊髓源星形胶质细胞sIL-6R基因表达抑制效果。结果:成功构建sIL-6R siRNA重组质粒真核表达载体,免疫组化染色、RT-PCR及Western blot检测结果证实sIL-6R siRNA能显著下调sIL-6R的表达。结论:sIL-6Rs iRNA重组质粒真核表达载体对脊髓损伤后sIL-6R表达有明显的抑制作用,为后续多靶点RNA干扰技术在脊髓损伤修复中的应用奠定了前期基础。Objective To construct a high interference efficiency vertor named RNA interference vector targeting interleukin 6 receptor （sIL-6R） gene, and research its effects on sIL-6R expressive suppression of spinal cord-derived astrocytes. Methods Three pairs of short hairpin RNA （short hairpin RNA, shRNA） oligonucleotide fragments targeting sIL-6R gene were designed, sIL-6R-shRNA recombinant plasmid eukaryofic expression vector was constructed, and transfected it into spinal cord astrocytes. Then, optimal screening sIL-6R siRNA on inhibiting expression of sIL-6R efficiency was taken. The effects of sIL-6R expressive suppression of spinal cord-derived astrocytes were detected with real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction （RT-PCR）, immunohistochemical staining and western blot. Results sIL-6R-shRNA recombinant plasmid eukaryotic expression vector was constructed successfully. The results of immunohistochemical staining, RT-PCR and western blot confirmed that the sIL-6R-gene expression could be effectively suppressed by the slL-6R-shRNA. Conclusion The sIL-6R-gene expression can be effectively suppressed by the sIL-6R-shRNA recombinant plasmid eukaryotic expression vector, paving the way for the application of the following multiple targets of RNA interference （RNAi） genetic treatment of repair of spinal cord injury.

关 键 词：RNA干扰 SIL-6R 脊髓损伤 星形胶质细胞 

分 类 号：R745.4[医药卫生—神经病学与精神病学]