Cu^(2+)对MC3T3-E1细胞增殖分化影响及对OPG、MEPE表达的影响

Effect of Cu^(2+) on the proliferation,differentiation of MC3T3-E1 and OPG,MEPEmRNA expression

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 detect Cu2 ＋ effect on osteoblast＇ s proliferation, differentiation and the expres- sion of OPG and MEPE. Method By using methods of 3- （4,5-dimethyhhiazol-2-yl） -2,5-diphenyltet-razolium bromide （ MTT）, alkaline phosphatase （ALP） activity assay, we studied the effect of 1 × 10（ -4 --6） mol/L Cu2＋ on the proliferation and differentiation of osteoblasts. By using method of RT - PCR to detect the expression level of OPG and MEPEmRNA in osteoblast, further study the molecular mechanisms of osteoblast proliferation, differentiation. Results the effect of 48 h and 96 h, 1×10^-6 mol/L Cu2＋ could markedly promote the proliferation of MC3T3 -E1 cells; the role of the 24 h, this con- centration has no effect on cell proliferation; 72 h, 1 ×10^-6mol/L Cu2＋ significantly inhibited cell prolif- eration; 1 × 10 -4mol/L,1 × 10^-6mol/LCu 2. on the proliferation of MC3T3 - E1 cells had no effect or in- hibit their proliferation. 1× 10-6mol/LCu2＋ in the role of 24 h, had no effect on cell differentiation, oth- er time can promote cell differentiation; 1 × 10-4mol/LCu2＋ can inhibit its differentiation, but 1 x 10-5 mol/LCu2＋ in the role of 96 h,can significantly promote the differentiation of MC3T3 -E1 cells. Agarose gel electrophoresis image can be seen 1 ×10 （-4--6）mol/L Cu2＋ acts on MC3T3 -E1 cells at 48 h, OPG and MEPEmRNA were expressed. Real time - PCR results show that the effect of 48 h of Cu2 ＋ on the os- teoblast, enhanced the expression of OPG and MEPE gene in osteoblast. Conclusion Effects of 48 h, 1 × 10-6 mol/L Cu2＋ can promote the proliferation and differentiation of MC3T3 -E1 cells,and its mecha- nism and OPG, MEPEmRNA related.