CAJ | 학술논문

背景：骨保护素和一氧化氮在防治骨质疏松方面有重要作用，但目前关于两者在抑制破骨细胞增殖分化方面的关系研究较少。 　　目的：验证不同剂量的骨保护素对破骨细胞内生成一氧化氮量及内皮型一氧化氮合酶活性的影响。 　　方法：用抗酒石酸酸性磷酸酶染色验证诱导生成的破骨细胞；将诱导生成的破骨细胞分成6个组，空白对照组不加任何试剂；阴性对照组培养液中加入培养液；骨保护素组分为4组分别加入10，25，50，75μg/L不同剂量的骨保护素试剂。采用Annexinv-FITC细胞凋亡检测试剂盒，利用流式细胞仪测定破骨细胞凋亡率；荧光定量PCR检测破骨细胞标志基因抗酒石酸酸性磷酸酶mRNA及蛋白激酶K mRNA 的表达量变化；一氧化氮检测试剂盒检测破骨细胞中内一氧化氮浓度；内皮型一氧化氮合成酶活力试剂盒检测破骨细胞内一氧化氮合酶的活力；骨保护素各组加入内皮细胞型一氧化氮合酶抑制剂；荧光定量PCR检测破骨细胞特异性酶抗酒石酸酸性磷酸酶 mRNA及蛋白激酶K mRNA表达量的变化。 　　结果与结论：①骨保护素可以抑制破骨细胞的分化生成并诱导其凋亡。②骨保护素的质量浓度与诱导生成的破骨细胞数量及其标志酶mRNA的表达量呈负相关，与破骨细胞凋亡率呈正相关。③骨保护素可以增加破骨细胞内一氧化氮的生成以及内皮型一氧化氮合酶活性的升高；骨保护素的质量浓度与破骨细胞生成的一氧化氮浓度及内皮型一氧化氮合酶活性呈正相关。④Raw264.7细胞在体外培养条件下，骨保护素与一氧化氮在抑制破骨细胞生成及促进其凋亡方面有协同作用，推测两者之间可能存在骨保护素/内皮型一氧化氮合酶/一氧化氮信号通路。
배경：골보호소화일양화담재방치골질소송방면유중요작용，단목전관우량자재억제파골세포증식분화방면적관계연구교소。 　　목적：험증불동제량적골보호소대파골세포내생성일양화담량급내피형일양화담합매활성적영향。 　　방법：용항주석산산성린산매염색험증유도생성적파골세포；장유도생성적파골세포분성6개조，공백대조조불가임하시제；음성대조조배양액중가입배양액；골보호소조분위4조분별가입10，25，50，75μg/L불동제량적골보호소시제。채용Annexinv-FITC세포조망검측시제합，이용류식세포의측정파골세포조망솔；형광정량PCR검측파골세포표지기인항주석산산성린산매mRNA급단백격매K mRNA 적표체량변화；일양화담검측시제합검측파골세포중내일양화담농도；내피형일양화담합성매활력시제합검측파골세포내일양화담합매적활력；골보호소각조가입내피세포형일양화담합매억제제；형광정량PCR검측파골세포특이성매항주석산산성린산매 mRNA급단백격매K mRNA표체량적변화。 　　결과여결론：①골보호소가이억제파골세포적분화생성병유도기조망。②골보호소적질량농도여유도생성적파골세포수량급기표지매mRNA적표체량정부상관，여파골세포조망솔정정상관。③골보호소가이증가파골세포내일양화담적생성이급내피형일양화담합매활성적승고；골보호소적질량농도여파골세포생성적일양화담농도급내피형일양화담합매활성정정상관。④Raw264.7세포재체외배양조건하，골보호소여일양화담재억제파골세포생성급촉진기조망방면유협동작용，추측량자지간가능존재골보호소/내피형일양화담합매/일양화담신호통로。
BACKGROUND:Osteoprotegerin and nitrogen monoxidum play a key role in the prevention and treatment of osteoporosis. However, the correlation of the two in inhibiting the proliferation and differentiation of osteoclasts remains unclear. OBJECTIVE:To observe the effect of different doses of osteoprotegerin on nitric oxide production and endothelial nitric oxide synthase activity in osteoclasts. METHODS:Tartrate resistant acid phophatase staining was used to test whether the induced cells are osteoclasts. Osteoclasts were divided into six groups:blank control group (no reagent);negative control group (DMEM);four osteoprotegerin groups (10, 25, 50, 75μg/L of osteoprotegerin). Annexinv-FITC kit and flow cytometry were used to test the apoptosis rate of osteoclasts. Real-time PCR was used to detect the expression of osteoclasts marker gene, TRAP mRNA and protein kinase K mRNA. Nitric oxide production and nitric oxide synthase activity were determined using the corresponding kits. Four osteoprotegerin groups were added with L-NAME, a kind of nitric oxide synthase inhibitor, to test the changes of the apoptosis rates of osteoclasts and the changes of the expression of TRAP mRNA and protein kinase K mRNA of osteoclasts. RESULTS AND CONCLUSION:Osteoprotegerin inhibited the differentiation of osteoclasts and induced the apoptosis. Osteoprotegerin concentration had a positive correlation with the apoptosis rate of osteoclasts, and a negative correlation with the numbers of osteoclasts and expression of marker gene TRAP mRNA and protein kinase K mRNA in osteoclasts. Osteoprotegerin boosted the nitric oxide production and endothelial nitric oxide synthase activity, and osteoprotegerin concentration was positively correlated to the nitric oxide production and endothelial nitric oxide synthase activity. After Raw264.7 cells were in vitro cultured, osteoprotegerin and nitric oxide play a synergic role in inhibiting osteoclasts production and promoting the apoptosis. We speculate that there is osteoprotegerin/endothelial nitric oxide synthase/nitric oxide signal pathway.