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目的观察Cu2+对小鼠成骨细胞(MC3T3-E1)增殖、分化以及OPG与MEPE表达。方法采用不同浓度1×10(-4～-6)mol/L Cu2+作用于MC3T3-E1细胞,分别在24、48、72、96 h后采用MTT法检测Cu2+对细胞增殖的影响;ALP(碱性磷酸酶)试剂盒检测细胞培养液中ALP活性;RT-PCR方法检测1×10(-4～-6)mol/L Cu2+作用下细胞中OPG和MEPE的mRNA表达水平,探讨MC3T3-E1细胞增殖分化的分子机制。结果作用48 h和96 h时,1×10-6mol/L Cu2+明显促进MC3T3-E1细胞增殖;作用24 h时,此浓度对细胞增殖无影响;作用72 h时,1×10-6mol/L Cu2+明显抑制细胞增殖;1×10-4mol/L、1×10-5mol/L的Cu2+对MC3T3-E1细胞增殖无影响或抑制其增殖。ALP试剂盒检测1×10-6mol/LCu2+在作用24 h时,对细胞分化无影响,其他时间均可促进细胞分化;1×10-4mol/L的Cu2+抑制细胞分化,1×10-5mol/LCu2+在作用96 h时,可显著促进MC3T3-E1细胞分化,其他时间对细胞分化无影响。琼脂糖凝胶电泳图片可见1×10(-4～-6)mol/LCu2+作用于MC3T3-E1细胞48 h时,OPG和MEPEmRNA均有表达。实时荧光定量PCR结果表明,在Cu2+浓度为1×10-6mol/L作用48 h时OPG与MEPEmRNA明显增加。结论作用48 h时,1×10-6mol/L Cu2+可以促进MC3T3-E1细胞增殖和分化,其机制与OPG、MEPEmRNA相关。
Objective To observe the proliferation and differentiation of mouse osteoblasts (MC3T3-E1) induced by Cu2 + and the expression of OPG and MEPE. METHODS: MC3T3-E1 cells were treated with 1 × 10-4 mol / L Cu2 + at different concentrations for 24, 48, 72 and 96 h, respectively. MTT assay was used to detect the effect of Cu2 + on cell proliferation. ALP The activity of ALP in cell culture medium was detected by RT-PCR. The mRNA expression levels of OPG and MEPE in cells treated with 1 × 10-4 mol / L Cu2 + were detected by RT-PCR. The expression of OPG and MEPE mRNA in MC3T3-E1 cells The molecular mechanism of proliferation and differentiation. Results After treated with 1 × 10-6 mol / L Cu2 + for 48 h and 96 h, the proliferation of MC3T3-E1 cells was significantly promoted. At 24 h, this concentration had no effect on cell proliferation. At 72 h, 1 × 10-6 mol / L Cu2 + significantly inhibited cell proliferation; 1 × 10-4mol / L, 1 × 10-5mol / L of Cu2 + had no effect on MC3T3-E1 cell proliferation or inhibited its proliferation. ALP kit detected 1 × 10-6mol / LCu2 + in the role of 24 h, no effect on cell differentiation, and other time can promote cell differentiation; 1 × 10-4mol / L of Cu2 + inhibition of cell differentiation, 1 × 10-5mol / LCu2 + could significantly promote the differentiation of MC3T3-E1 cells at 96 h, and had no effect on cell differentiation at other times. The results of agarose gel electrophoresis showed that OPG and MEPE mRNA were all expressed in MC3T3-E1 cells treated with 1 × 10 (-4 ~ -6) mol / L Cu2 + for 48 h. Real-time PCR results showed that OPG and MEPE mRNA increased significantly at a concentration of 1 × 10-6mol / L Cu2 + for 48 h. Conclusions At 48 h, 1 × 10-6 mol / L Cu2 + can promote the proliferation and differentiation of MC3T3-E1 cells. The mechanism is related to OPG and MEPE mRNA.