联合应用骨保护素和阿伦膦酸钠对破骨前体细胞分化的影响

Effects of combination of bone protectin and alendronate on differentiation of osteoclast precursors

目的:探讨联合应用重组人骨保护素活性片断和阿伦膦酸钠对破骨细胞前体细胞(RAW264.7)分化的抑制效果,并探讨二者是否具有抑制破骨细胞前体细胞(RAW264.7)分化的协同作用。方法:实验于2004-06/12在解放军总医院骨科研究所完成。酶消化法获取新生小鼠颅骨成骨细胞,与破骨细胞前体细胞(RAW264.7)按照4:1比例混合,培养于96孔板中和6孔板骨磨片体系中。分别使用1×10-5g/L重组人骨保护素活性片断、10mol/L阿伦膦酸钠、1×10-5g/L重组人骨保护素活性片断+10mol/L阿伦膦酸钠作用于96孔板和6孔板混合细胞体系,并设空白对照。9d后,观察破骨细胞数目和形态,计算单位面积内重酒石酸盐抗酸性磷酸酶染色阳性细胞个数,并行骨磨片吸收陷窝计数。统计学分析评价其抑制破骨细胞分化效果。结果:①破骨细胞常规形态及重酒石酸盐抗酸性磷酸酶染色阳性细胞计数:小鼠成骨细胞与RAW264.7混合培养并加入药物后9d,空白对照组出现大量多核成熟破骨细胞,重酒石酸盐抗酸性磷酸酶染色证实为成熟破骨细胞。②骨磨片吸收陷窝形态及计数:与空白对照组相比,其余各组破骨细胞重酒石酸盐抗酸性磷酸酶染色阳性细胞个数,骨磨片吸收陷窝计数均明显减少,以1×10-5g/L重组人骨保护素活性片断+10mol/L阿伦膦酸钠组减少最为显著。③重组人骨保护素活性片断组与阿伦膦酸钠组析因设计的方差分析:加药后9d重组人骨保护素活性片断与阿伦膦酸钠存在交互作用(F=19.878,623.57;P<0.05)。结论:联合应用重组人骨保护素活性片断和阿伦膦酸钠可以发挥交互协同作用,更有效地抑制RAW264.7向成熟破骨细胞的分化。

AIM： To study the inhibitive effect of combination of rhOPG-Fc and alendronate （ALN） on osteoclasts precursor （,RAW264.7） differentiation, and explore if they had the synergistic action on inhibiting osteoclasts precursor （RAW264.7） differentiation. METHODS： The experiment was performed at the Institute of Department of Orthopaedics, General Hospital of Chinese PLA between June and December 2004. Osteoblasts were got from new-born mouse skeletal bone with enzymatic digestion method, and mixed with osteoclasts precursor （RAW264.7） according to the proportion of 4 to 1. They were cultured in the 96 well plate and 6 well plate cortical bone sliees system. The 1×10^-5g/L rhOPG-Fc, 10 mol/L ALN and 1×10^-5g/L rhOPG-Fc＋10 mol/L ALN were added to 96 well plate and 6 well plate co-culture systems, and the empty control group was set up. After 9 days, counting and formation of osteoclasts were observed, and tartrate resistance acid phosphatase （TRAP） stain positive cells quantity was calculated in unit area, and the cortical bone resorption pit counting Were preformed. Its differentiation effect on inhibiting osteoclast was assessed with statistics analysis. RESULTS： ① The routine formation of osteoclasts and TRAP stain positive cells counting： After culturing for 9 days of combination of osteoblasts and RAW264.7 and adding the drugs, many muhi-nucleared mature osteoclasts verified by the TRAP stain were found in empty control group. ② Cortical bone resorption pit formation and counting： Compared with the empty control group, the TRAP stain positive cells counting and cortical bone resorption pit counting both decreased significantly in other groups. It reduced most obviously in the 1×10^-5g/L rhOPG-Fc＋10 mol/L ALN group. ③ Factorial design analysis of variance in the rhOPG-Fc group and the ALN group： There was interaction between rhOPG-Fc and ALN after administration for 9 days （F=19.878, 623.57; P 〈 0.05）. CONCLUSION： The combination of rhOPG-Fc and ALN can bring exchanged synergistic action, and more effectively inhibit RAW264.7 differentiation into mature osteoclasts.