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目的构建及鉴定小鼠干扰素调节因子8(IRF8)重组腺病毒Ad-IRF8,并观察过表达的IRF8对小鼠可溶性核因子κB受体活化因子配体(sRANKL)诱导的RAW264.7细胞分化的影响。方法 PCR扩增IRF8,亚克隆到穿梭载体pAdTrack-CMV中,PmeⅠ线性化后与骨架载体pAdEasy-1在大肠杆菌BJ5183中同源重组获得重组腺病毒载体pAd-IRF8。pAd-IRF8经PacⅠ线性化后转染AD293细胞,包装出重组腺病毒(Ad-IRF8)。Ad-IRF8感染小鼠RAW264.7细胞,经半定量反转录PCR(RT-PCR)和Western blot法检测IRF8的过表达,通过抗酒石酸酸性磷酸酶(TRAP)染色检测IRF8对sRANKL诱导的RAW264.7细胞分化的影响。结果成功构建携带IRF8基因的穿梭载体pAdTrack-IRF8-CMV和重组载体pAd-IRF8,并且在AD293细胞中成功包装出重组腺病毒Ad-IRF8。半定量RT-PCR和Western blot法证实IRF8在RAW264.7细胞中有过表达,TRAP染色结果显示过表达的IRF8有效抑制RAW264.7细胞向破骨样细胞分化。结论成功构建重组腺病毒Ad-IRF8,在RAW264.7细胞中检测到了IRF8过表达,并能有效抑制RAW264.7细胞向破骨样细胞分化。
Objective To construct and identify the recombinant adenovirus Ad-IRF8 expressing mouse interferon regulatory factor 8 (IRF8) and observe the effect of overexpressed IRF8 on the differentiation of RAW264.7 cells induced by sRANKL (mouse nuclear factor-kappa B receptor activator ligand) Impact. Methods IRF8 was amplified by PCR and subcloned into the shuttle vector pAdTrack-CMV. After linearization with PmeⅠ, the recombinant adenovirus vector pAd-IRF8 was obtained by homologous recombination with the backbone vector pAdEasy-1 in E. coli BJ5183. pAd-IRF8 was linearized with PacI and then transfected into AD293 cells to package recombinant adenovirus (Ad-IRF8). The RAW264.7 cells were infected with Ad-IRF8 and the over-expression of IRF8 was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The effect of IRF8 on sRANKL-induced RAW264 .7 cell differentiation. Results The shuttle vector pAdTrack-IRF8-CMV carrying the IRF8 gene and the recombinant vector pAd-IRF8 were successfully constructed and the recombinant adenovirus Ad-IRF8 was successfully packaged in AD293 cells. Semi-quantitative RT-PCR and Western blot confirmed that IRF8 was overexpressed in RAW264.7 cells. TRAP staining showed that overexpression of IRF8 effectively inhibited the differentiation of RAW264.7 cells into osteoclast-like cells. Conclusion The recombinant adenovirus Ad-IRF8 was successfully constructed and IRF8 overexpression was detected in RAW264.7 cells, which can effectively inhibit the differentiation of RAW264.7 cells into osteoclast-like cells.