过表达小鼠干扰素调节因子8抑制RAW264.7细胞向破骨样细胞的分化

Overexpression of mouse IRF8 inhibits the differentiation of RAW264.7 cells into osteoclast-like cells

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 recombinant adenovirus Ad-IRF8 carrying mouse interferon regulatory factor-8 （IRF8） and observe the influence of the over-expressed IRF8 on the differentiation of osteoclast precursor cells （RAW264.7） induced by soluble receptor activator of nuclear factor KB ligand （sRANKL）. Methods Mouse IRF8 gene amplified by PCR was subcloned into the shuttle vector pAdTrack-CMV. The vector pAdTrack-IRF8-CMV was linearized by Pme I, followed by homologous recombination with bone vector pAdEasy-1 in E. coli BJ5183. Recombinant adenovirus vector pAd-IRF8 was obtained and linearized by Pac I, and then transfected into AD293 cells. The recombinant adenovirus Ad-IRF8 was harvested after packaged in AD293 cells. RAW264.7 cells were infected by Ad-IRFS. The overexpression of IRF8 was verified with semi-quantitative reverse transcription-PCR （RT-PCR） and Western blotting. The influence of the overexpression of IRF8 on sRANKL-induced differentiation of RAW264.7 cells was detected by tartrate-resistant acid phosphatase （TRAP） staining. Results The shuttle vector pAdTrack-IRF8-CMV and recombinant vector pAd-IRF8 were successfully constructed. The recombinant adenovirus Ad-IRF8 was also successfully packaged in AD293 cells. The overexpression of IRF8 in RAW264.7 cells was confirmed by RT-PCR and Western blotting. TRAP staining showed that the 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 the overexpression of IRF8 was also detected in RAW264.7 cells. The overexpressed IRF8 can effectively inhibit the differentiation of RAW264.7 cells into osteoclast-like cells.