骨保护素对破骨细胞分化和骨吸收活性的抑制作用

背景骨保护素(osteoprotegerin,OPG)可抑制破骨细胞的分化,抑制成熟破骨细胞的骨吸收活性并诱导其凋亡,在骨质疏松、类风湿性关节炎、癌症骨转移的领域有潜在的应用价值.目的鉴定在CHO细胞中表达的人骨保护素生物学活性.设计随机对照的实验研究.地点和对象实验在解放军第四军医大学西京医院骨科研究所完成,研究对象人骨肉瘤细胞株MG63、中国仓鼠CHO细胞株、克隆质粒pUC19及真核表达质粒pcDNA3为本室保存,12只6～8周龄BABL/c雄性小鼠由本校动物中心提供.干预RT-PCR法获得人OPG编码区cDNA并构建真核表达载体,在脂质体介导下转染CHO细胞,经Western blot鉴定筛选稳定表达OPG的细胞系.获取含有OPG蛋白的条件培养基浓缩液,实验组体外培养的鼠破骨细胞培养基中加入含有OPG蛋白的条件培养基浓缩液.对照组1只加入转染pcDNA3空载体的CHO细胞培养基的浓缩液.对照组2只加入完全培养基.主要观察指标观察人OPG对破骨细胞的分化和骨吸收的影响.结果转染人OPG编码区基因的CHO细胞能分泌表达OPG.小鼠骨髓细胞在1α,25(OH)2D3(1×10-8mol/L)的存在下可稳定分化出具有骨吸收功能的破骨细胞样细胞,在该培养体系中加入含有OPG的CHO细胞培养上清浓缩液,TRAP染色阳性细胞数明显减少(t=5.547,P＜0.01),骨吸收陷窝的数量显著减少(t=3.409,P＜0.01).结论人骨保护素可在CHO细胞中分泌表达,并对体外培养状态下的破骨细胞的分化和骨吸收功能有抑制作用.

Inhibition effect of osteoprotegerin on the differentiation and the activity of bone absorption of osteoclast

BACKGROUND: Osteoprotegerin(OPG) can inhibit the differentiation of osteoclast, inhibit the activity of bone absorption of mature osteoclast and induce its apoptosis, whicb has potential merit in the fields of osteoporosis,rheumatoid arthritis, and cancerous bone metastasis.OBJECTIVE: To identify the biological activity of human OPG expressed in CHO cells.DESIGN: A randomized controlled experimental study was conducted.SETTING and PARTICIPANTS: The experiment was completed in the Institute of Orthopaedic Surgery, Xijing Hospital, Fourth Military Medical University. The subjects were human osteosarcoma cell strain MG63, Chinese hamster CHO cell strain, clonal plasmid pUC19 and eukaryon-expressed plasmid pcDNA3 were stored by our department. Twelve male BABL/c mice aged 6-8 weeks old were obtained from Center for Experimental Animals of our university.INTERVENTIONS: cDNA in human OPG code area was acquired through RT-PCR method and the eukaryon-expressed carrier was establishezd as well,and then CHO cells were transfected under the mediation of liposome. Cell line that could stably express OPC was scanned by Western blot. Enriched liquid of conditional culture medium containing OPG protein was obtained. Experiment group: enriched liquid of conditional culture medium containing OPG protein was added into mice osteoclast culture medium in vitro. Control group: emiched liquid of CHO culture medium containing transfected pcDNA3 empty carrier was added into control group 1 and complete culture nedium was added into control group 2.MAIN OUTCOME MEASURES: The effects of human OPG on the differentiation and bone absorption of osteoclast.RESULTS: CHO cells that transfected by human OPG code area genes could secrete and express OPG. Myelocyte in mice could stably differentiate osteoclast-like cell that has the function of bone absorption under the existence of lα, 25(OH) 2D3(1 × 10-8 mol/L) . TRAP stained positive cells significantly decreased in the culture system, which was added by enriched supernatant of CHO culture solution containing OPG( t = 5. 547, P ＜ 0.01 ), and the numbers of bone absorption lacuna significantly decreased as well( t =3.409, P ＜0.01).CONCLUSION: Human OPG could be secreted and expressed in CHOcells, which has inhibitive reactions on the differentiation and bone absorption of osteoclast under the condition of culture in vitro.