重组人骨靶向干扰素γ的骨靶向性和对巨噬细胞向破骨细胞分化的影响

The Bone Targeting of Recombinant Human Interferon Gamma and its Effect on Differentiation of Macrophages into Osteoclasts

目的对重组人骨靶向干扰素γ(rhIFNγ-D10)进行体外骨靶向性和促进巨噬细胞向破骨细胞分化的活性分析。方法采用羟基磷灰石体外结合实验分析钙亲和力,表征目的蛋白靶向骨组织能力。分别测定重组人骨靶向干扰素γ(rhIFNγ-D10)和非骨靶向性重组人干扰素γ(rhIFNγ)体外结合羟基磷灰石的能力并加以比较,分析天冬氨酸寡肽对干扰素γ骨靶向性的影响。用RANKL、RANKL+rhIFNγ-D10、RANKL+rhIFNγ、rhIFNγ-D10和rhIFNγ分别诱导小鼠巨噬细胞RAW264.7向破骨细胞分化,抗酒石酸酸性磷酸酶(TRAP)染色法评价rhIFNγ-D10对破骨细胞分化形成的影响。结果rhIFNγ-D10体外结合羟基磷灰石的能力明显高于rhIFNγ,说明天冬氨酸寡肽有助于将干扰素γ靶向骨组织。与RANKL对照组相比,RANKL+rhIFNγ-D10组和RANKL+rhIFNγ组破骨细胞分化染色细胞明显减少,说明在RANKL诱导破骨细胞分化条件下,rhIFNγ-D10和rhIFNγ对破骨细胞分化有抑制作用。结论采用大肠杆菌表达的rhIFNγ-D10具有骨靶向性,但未显示预期的促进小鼠巨噬细胞向破骨细胞分化的作用,相反表现出一定的抑制作用,其分子机制有待进一步研究。

Objective Recombinant human interferon gamma tagged with an acidic oligopeptide(rhIFNγ-D10)has been expressed and purified from E.coli,but its bone-targeting and effect on osteoclastogenesis remained to be identified.Methods The C-terminal of interferon gamma was tagged with an acidic oligopeptide(a ten residue stretch of Asp),and the biochemical properties of the purified tagged and untagged interferon gamma derived from E.coli including its bonetargeting and effect on osteoclastogenesis were compared.Hydroxyapatite was used in vitro bone-targeting affinity experiments to compare the tagged interferon gamma to the untagged interferon gamma.In addition,RANKL,RANKL+rhIFNγ-D10,RANKL+rhIFNγ,rhIFNγ-D10 and rhIFNγwere used to induce RAW264.7 cells to differentiate into osteoclasts,respectively.The effect of the tagged and the untagged interferon gamma on osteoclastogenesis were evaluated by tartrate-resistant acid phosphatase(TRAP)staining.Results In vitro affinity experiments showed the Asp-tagged interferon gamma had 3-fold higher affinity for hydroxyapatite compared to the untagged interferon gamma.Both the tagged and untagged interferon gamma could not significantly increase the number of osteoclasts by TRAP.In contrast,Both of them could significantly inhibit the differentiation of osteoclasts induced by RANKL.Conclusion The tagged interferon gamma with a ten Asp acidic oligopeptide is of good bone-targeting,but it can not increase the differentiation of macrophages into osteclasts.