吗啡对体外培养正常女性成骨细胞的增殖及BGP mRNA表达的影响

EFFECT OF MORPHINE ON PROLIFERATION AND BGP mRNA EXPRESSION OF CULTURED FEMALE OSTEOBLASTS IN VITRO

目的:观察阿片类药物是否对体外培养正常女性成骨细胞增殖有影响。方法:用胰酶-胶原酶消化法从正常女性松质骨中分离培养成骨细胞,用茜素红染色法、改良Gomonri钙钴法鉴定成骨细胞;制备不同浓度的吗啡及其拮抗剂纳洛酮,作用于体外培养的成人正常成骨细胞上,通过四甲基偶氮唑盐(MTT)比色法检测吗啡对其的影响;通过RT-PCR法半定量分析不同浓度的吗啡及其拮抗剂纳洛酮对成骨细胞骨钙素(BGP mRNA)表达的影响。结果:用酶消化法分离的人成骨细胞,在体外培养时可合成碱性磷酸酶(ALP)、形成钙化结节。不同浓度的吗啡组与对照组OD490(optical density,OD)值相比差异有显著性(P<0.001);1×10-7mol·L-1、1×10-6mol·L-1、1×10-5mol·L-1和1×10-4mol·L-1吗啡组的细胞增殖速度依次降低(P<0.001);而吗啡(10-5mol·L-1)+纳洛酮(10-5mol·L-1)组与对照组OD490值相比差异不明显(P>0.05)。吗啡作用24 h呈剂量依赖性抑制BGPmRNA的表达。结论:用酶消化法进行人成骨细胞培养,简捷易行,可培养大量高纯度人成骨细胞供研究使用;外源性阿片类药物吗啡可直接抑制人成骨细胞的增殖。阿片类物质可以抑制成骨细胞内BGP mRNA的表达。

Objective：To evaluate the effect of morphine on proliferation of normal female cultured osteoblasts cell in vitro.Methods：The human osteoblasts（HOBs）were isolated from the trabecular bone by trypsin and collagenase digestion.Its functions were identified by alizarin red staining and modified Gomonri method.The effects of morphine on proliferation of cultured HOBs in vitro and morphine and naloxone with different concentrations on BGP mRNA expression were evaluated with MTT assay,and RT-PCR semi-quantity analysis,respectively.Results：The HOBs purified by enzyme digestion in vitro had a normal function showing synthesized alkaline phosphatase（ALP）and forming calcified nodules as observed in vivo,and significant difference in optical density（OD490） was found between morphine group with different concentrations and the control group（P〈0.001）,but no significant difference showed in the morphine（1×10^-5mol·L^-1＋ naloxone（1×10^-5mol·L^-1group,compared with the control group（P〉0.05）.The speed of cell proliferation degraded as the morphine concentration increased（1×10^-7 mol·L^-11×10^-6mol·L^-11×10^-5mol·L^-11×10-4 mol·L^-1（P〈0.001）.BGP mRNA expression was depressed by morphine（at 24 h）in the patern of dose-dependence.Conclusion：Enzyme digestion is a good and easy method for purifying plentiful HOBs.Opioids can directly inhibit HOBs proliferation.