蛋白酶体抑制剂MG-132阻碍破骨细胞分化成熟的研究

Inhibitive effect of proteasome inhibitor MG-132 on osteoclast

目的探讨蛋白酶抑制剂MG-132对破骨细胞分化成熟过程的影响。方法分离、培养新生兔四肢长骨破骨细胞,实验分4组：对照组、MG-132 0.001 mmol/L培养组、0.01 mmol/L培养组及0.1 mmol/L培养组。于培养第2、5、8 d取细胞玻片、皮质骨片进行HE、甲苯胺蓝、抗酒石酸酸性磷酸酶（TRAP）染色,光镜下观察染色阳性细胞和骨片吸收陷窝,扫描电镜下观察吸收陷窝形态。结果（1）与对照组（第5天：46.8±3.4,第8天：34.6±4.1）比较,MG-132 0.001 mmol/L培养组、0.01 mmol/L培养组及0.1 mmol/L培养组破骨细胞计数在培养5 d后明显降低,分别为40.4±4.6（P〈0.05）、38.4±3.9（P〈0.05）、31.9±5.6（P〈0.01）;（2）破骨细胞计数与蛋白酶抑制剂MG-132浓度呈负相关（r=-0.342,P〈0.01）;（3）与对照组比较,MG-132 0.01 mmol/L培养组及0.1 mmol/L培养组骨片骨陷窝计数在培养第2、5、8天时,均明显减少（P〈0.05）,0.001 mmol/L培养组与对照组比较差异无统计学意义。结论蛋白酶抑制剂MG-132可阻碍破骨细胞的分化成熟,抑制其蚀骨作用,并呈剂量依赖性。

Objective To observe the effect of proteasome inhibitor MG-132 on the differentiation and maturation of osteoclast.Methods The osteoclasts were isolated from the long bones of neonatal New Zealand S rabbits,and were cultured on the glass cover slips（10 mm×10 mm）and bone slices（10mm×10 mm×50μm）.The plates of cultures were divided into four groups：control group,the group cocultured with 0.001 mmol/L MG-132,0.01 mmol/L MG-132 and 0.1 mmol/L MG-132.The glass cover slips and bone slices were stained by HE,toluidine blue and tartrate-resistant acid phosphatase（TRAP）at 2,5,8 days,and TRAP positive multi-nucleated cells and bone resorption lacunes were counted.Scanning electron microscopy was used to observe the pits of bone resorption.Osteoclasts were identified as multi-nucleated TRAP-positive cells.Results （1）Compare to the control group（5 d：46.8±3.4,8 d：34.6±4.1）,the number of osteoclast was significantly decreased in MG-132 groups of 0.001 mmol/L（40.4±4.6,P〈0.05）,0.01 mmol/L （38.4±3.9,P〈0.01）and 0.1 mmol/L（31.9±5.6,P〈0.01）at the fifth coculture day;（2） The number of osteoclast was negatively associated with the concentration of MG-132（r=-0.342,P〈0.01）;（3）Since the second day,the number of bone resorption laeunes in group 0.01 mmol/L and group 0.1 mmol/L of MG-132 were significantly lower than those in control group（all P〈0.05）. There was no significant difference in the number of bone resorption lacunes between group 0.001 mmol/L of MG-132 and control group（P〉0.05）.Conclusions MG-132 could hinder the differentiation and maturation of osteoclast,and inhibit its bone resorption function in a dose dependent manner.