【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。更多还原

【Abstract】 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 eukaryotic 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 sIL-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.更多还原