骨保护素体外抑制小鼠单核巨噬细胞成熟分化的实验研究

Study on osteoprotegerin in inhibitation of mouse osteoclast precursor cell differentiation

目的研究骨保护素(Osteoprotegerin,OPG)抑制核因子NF-κB受体活化因子配体(Receptoractivator of nuclear kappa B ligand,RANKL)诱导小鼠单核细胞RAW264.7成熟分化而导致的溶骨效应。方法 50 ng/mL RANKL诱导RAW264.7细胞1 d后,加入100 ng/mL OPG(实验组,即OPG+RANKL组)或不加入OPG(对照组,即RANKL组)分别培养7 d和9 d,经细胞形态学观察其变化,抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)染色法观察TRAP阳性多核细胞,扫描电镜下观察在骨片上的破骨细胞所致的骨吸收陷窝形成情况。结果对照组培养7 d时,在倒置相差显微镜、透射电镜、光镜下可见细胞形状为椭圆形或不规则形,胞体明显较RAW264.7细胞增大,胞核多为6～10个,扫描电镜下还可见大量伪足形成,而实验组培养7 d后,细胞形状多为圆形,且扫描电镜下未见明显伪足形成;对照组9 d时可见大量TRAP染色阳性的多核巨细胞(含3个或3个以上的细胞核),而实验组中TRAP染色阳性的多核破骨细胞偶见多核巨细胞,培养9 d时很难找到多核巨细胞;仅用RANKL诱导RAW264.7细胞分化7 d时,对照组中破骨细胞表面可见大量伪足伸出,并形成明显的骨吸收陷窝,实验组中破骨细胞见少许伪足突出,不能看到明显的骨陷窝形成。结论单用50 ng/mL RANKL体外连续诱导RAW264.7细胞7 d时,可以促进成熟的破骨细胞显著分化。100 ng/mL OPG培养9 d能有效地抑制破骨细胞的分化,减少破骨细胞的骨吸收效应。

Objective To observe whether OPG is effective in inhibiting the effect that RANKL induce RAW264.7 ceils to differentiation to mature osteoclast. Methods RAW264. 7 cells were solely treated with 50 ng/ml RANKL for 1 day, which then were divided into two groups： the one is an OPG group involving 100 ng/ml OPG and 50 ng/ml RANKL, the other is a control group only containing 50 ng/ml RANKL. After the period between 7 and 9 d, cells morphological changes can be investigated by Inverted Phase Control Microscope （IPCM） , Transmission Electron Microscope （TEM） , Scanning Electron Microscope （SEM） and Light Microscope respectively; Furthermore, Staining TRAP-positive multinucleated cells can be detected by IPCM;The resorption pits of bone slices were indicated through SEM. Results It is very clear that the sharp of RAW264.7 cells became oval or irregular, and their bodies go bigger significantly including 6 up to 10 nucleus in control group by means of IPCM, TEM and Light Microscope. Interestingly, we also have found that there were considerable pseudopodia-like protrusions attaching to the surface of these cells by Scanning Electron Microscopy in the control group. By contrast, the research result is polar in OPG group.The outcome of experiment has further demonstrated that RAW264.7 ceils treated by RANKL in consecutive 7 days performed more highly dramatic numbers of staining TRAP-positive multinucleated osteoelasts （more than 3 nucleus） in control group than corresponding OPG group; Finally, in terms of the number of bone resorption formation, control group has overwhelmingly outweighed than the OPG group. Conclusion RAW264. 7 cells could be successfully induced differentiation to mature osteoclasts on the condition of single 50ng/ml RANKL, especially in excess of 7 d. The interference of 100 ng/ml OPG in 9 days could be extremely beneficial to inhibit the process in osteoelasts differentiation and reduce the effect on bone resorption in an effeetive approach.