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利用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色和矿化结节染色及定量分析,研究了Cu2+和Cu+对原代培养的成骨细胞增殖、分化及钙化的影响。结果显示:Cu2+(1×10-9～1×10-6 mol.L-1)促进成骨细胞增殖,随时间延长,促进作用变弱。Cu+(1×10-7～1×10-5 mol.L-1)抑制成骨细胞增殖,随时间延长,浓度为1×10-6 mol.L-1的Cu+为促进作用,其余浓度则没有影响。对于成骨细胞分化,Cu2+和Cu+表现出相似的影响,浓度为1×10-9和1×10-6 mol.L-1时均促进成骨细胞分化,而当浓度为1×10-7和1×10-5mol.L-1时,则抑制成骨细胞分化,随作用时间延长,大多数浓度均表现为促进作用。测试浓度下的Cu2+和Cu+均对成骨细胞向脂肪细胞的横向分化表现为促进效应。对矿化功能的影响,1×10-5mol.L-1的Cu2+和Cu+表现出显著的抑制效应,但随浓度降低,抑制效应变弱。1×10-7 mol.L-1的Cu2+促进成骨细胞矿化结节的形成。结果提示:作用浓度、作用时间及铜离子的价态都是影响Cu2+和Cu+生物效应转变(从毒性到活性,从损伤到保护,从下调到上调)的关键因素。
The effects of Cu2 + and Cu + on primary cultured osteoblast proliferation and differentiation and the effects of Cu + on osteoblasts proliferation and differentiation were investigated by MTT assay, alkaline phosphatase (ALP) specific activity assay, oil red O staining and mineralized nodule staining. The impact of calcification. The results showed that Cu2 + (1 × 10-9 ~ 1 × 10-6 mol·L-1) could promote the proliferation of osteoblasts. With the extension of time, the promotion effect was weaker. Cu + (1 × 10-7 ~ 1 × 10-5 mol·L-1) inhibited the proliferation of osteoblasts. With time prolonging, the concentration of Cu + was 1 × 10-6 mol·L-1, No effect. For osteoblast differentiation, Cu2 + and Cu + showed similar effects. At concentrations of 1 × 10-9 and 1 × 10-6 mol·L-1, all promoted osteoblast differentiation. When the concentration was 1 × 10-7 And 1 × 10-5mol.L-1, the inhibition of osteoblast differentiation, with the prolonged role, most of the concentration showed a promotion. Both Cu2 + and Cu + at the test concentration showed a promoting effect on the transverse differentiation of osteoblasts to adipocytes. On the mineralization function, 1 × 10-5mol.L-1 of Cu2 + and Cu + showed a significant inhibitory effect, but with the concentration decreased, the inhibitory effect weakened. Cu2 + of 1 × 10-7 mol·L-1 promoted the formation of mineralized nodules in osteoblasts. The results suggest that both the concentration of action, the duration of action and the valence of copper ions are the key factors affecting the biological effects of Cu2 + and Cu + (from toxicity to activity, from injury to protection, down-regulation to up-regulation).