降钙素对破骨细胞增殖和凋亡的调节作用

Effect of calcitonin on the proliferation and apoptosis of osteoclasts

目的:降钙素对破骨细胞作用的研究以往主要集中在对破骨细胞骨吸收功能的调节上,实验通过观察不同浓度的降钙素对体外培养破骨细胞增殖及凋亡率的影响,进一步探讨降钙素对破骨细胞的作用机制。方法:实验于2006-08/2007-06在郑州大学医学院药理实验室完成。应用1dSD大鼠乳鼠,采用骨髓诱导法体外培养破骨细胞,并接种于盖玻片和骨磨片上。培养液中分别加入0,10-12,10-10,10-9,10-8mol/L的降钙素作用于大鼠破骨细胞,0mol/L为对照组。以抗酒石酸酸性磷酸酶(TRAP)染色法证实培养的破骨细胞,并于培养48h后观察破骨细胞数目、形态的变化;应用流式细胞仪检测不同浓度的降钙素对破骨细胞凋亡率的影响。结果:①破骨细胞的一般形态:破骨细胞较其他细胞大,形态不规则者,呈油煎蛋形、长条形、腊肠形或漏斗形等,细胞内可见几个至几十个核不等。抗酒石酸酸性磷酸酶活性部分为酒红色,颗粒状。②抗酒石酸酸性磷酸酶阳性细胞数:不同浓度降钙素培养液作用48h后,对盖玻片上的阳性细胞计数发现,破骨细胞数目随着降钙素浓度增加而减少(P<0.05),并且降钙素对破骨细胞增殖的抑制作用呈剂量依赖效应。③破骨细胞凋亡率:不同浓度的降钙素均有增加破骨细胞凋亡率的作用,且呈剂量依赖效应,10-10mol/L以上浓度的降钙素明显促进破骨细胞的凋亡效率(P<0.05)。结论:降钙素抑制破骨细胞的增殖,促进破骨细胞骨凋亡,从而抑制破骨细胞的骨吸收功能,并呈剂量相关性。

AIM： Previous researches concerning the effect on osteoclasts by calcitonin mainly focus on the regulation of bone resorption. This study was designed to observe the effects of different concentrations calcitonin on proliferation and apoptosis of osteoclasts cultured in vitro, so as to explore the action mechanism of calcitonin on osterclasts. METHODS： The study was carried out in the Pharmaceutical Laboratory of Pharmaceutical Science School of Zhengzhou University from August 2006 to June 2007. Osteoclasts were cultured from a one-day neonatal SD rat by bone marrow induction method, and incubated on cover slip and bone sheet with different concentrations of calcitonin （0,10^-12,10^-10,10^-9.10^-8 mol/L）, while 0 mol/L calcitonin was taken as control. The cultured osteoclasts were identified by the staining of tartrate-resistance acid phosphatase （TRAP） After 48 hours of the culture, the number and morphology of the osteoclasts were detected. The percentage of apoptic osteoclasts was measured with flow cytometry. RESULTS： Osteoclasts were bigger than other cells and had irregular shapes, showing frying egg, sliver, allanto-shape or infundibulate. Several or decades of nucleus were seen in cells. The cells were stained in red by TRAP, After cultured for 48 hours, the osteoclasts were decreased with increasing calcitonin concentrations （P 〈 0.05）, and this effect exhibited a dose-dependent manner.Calcitonin in the concentration from 10^-12 to 10^-8 mol/L could enhance the apoptosis of osteoclasts in a dose-dependent manner. The calcitonin over 10^-l0 mol/L significantly promoted the apoptosis of osteoclasts （P 〈 0.05）. CONCLUSION： With a dose-dependent manner, calcitonin inhibits osteoclasts proliferation and induces apoptosis of osteoctasts, while inhibits the bone resorption.