Cu2+和Cu+对原代培养的小鼠成骨细胞增殖、分化和钙化的影响

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^-5 mol·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^-5 mol·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。

Effects of Cu2+ and Cu+ on the Proliferation, Differentiation and Calcification of Primary Mouse Osteoblasts in vitro

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, oil red O assays and alizarin red-S (ARS) stain were employed to evaluate the effects of Cu²⁺ and Cu⁺ on proliferation, differentiation and calcification of primary osteoblasts (OBs) in vitro. The results showed that Cu²⁺ (1×10^-9~1×10^-6 mol·L^-1) promoted the proliferation of OBs for 48 h, and the promotive effect turned to weaken with prolonging incubation time. Cu⁺(1×10^-7~1×10^-5 mol·L^-1) inhibited the proliferation of OBs for 48 h, but turned to promote the proliferation of OBs at a concentration of 1×10^-6 mol·L^-1, had no effect on proliferation of OBs at other concentrations for 72 h. Both Cu²⁺ and Cu⁺ had similar effects on the differentiation of OBs, they promoted the differentiation of OBs at concentrations of 1×10^-9 and 1×10^-6 mol·L^-1 and inhibited the differentiation at concentrations of 1×10^-7 and 1×10^-5 mol·L^-1 for 48 h, promoted the differentiation at most concentrations for 72 h. Both Cu²⁺ and Cu⁺ promoted the adipocytic transdifferentiation of OBs at all tested concentrations. Both Cu²⁺ and Cu+ (1×10^-5 mol·L^-1) obviously inhibited the formation of mineralized matrix nodules of OBs, the inhibitory effect turned to weaken with decreasing concentration, Cu²⁺ promoted the formation of mineralized matrix nodules of OBs at a concentration of 1×10^-7 mol·L^-1. The results suggest that concentration, culture time and valence state of copper ion are key factors for switching the biological effects of Cu²⁺ and Cu⁺ from toxicity to activity, from damage to protection, or from down-regulation to up-regulation.