磷酸肌酸对小鼠前成骨细胞MC3T3-E1增殖、分化及矿化的影响

Phosphocreatine stimulates proliferation,differentiation and mineralization in mouse osteoblast-like MC3T3-E1 cells

目的探讨磷酸肌酸(phosphocreatine,PCr)对体外培养的小鼠前成骨细胞MC3T3-E1的增殖、成骨分化及矿化的影响。方法在体外培养的MC3T3-E1细胞中,分别加入含不同浓度(5,10,20 mmol·L^(-1))的PCr进行培养,并以不给予PCr药物处理的组为空白对照组。采用MTT法检测MC3T3-E1细胞的增殖情况;使用碱性磷酸酶(alkaline phosphatase,ALP)活性试剂盒检测MC3T3-E1细胞内ALP的活性;采用Western Blot法检测MC3T3-E1细胞内ALP、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)的蛋白表达情况;利用茜素红染色法检测MC3T3-E1细胞形成矿化的能力。结果 PCr可以促进MC3T3-E1细胞的增殖且具时间依赖性,当处理12 h时,PCr对细胞无明显促增殖作用(P>0.05);然而当处理48 h时,PCr(5,10,20mmol·L^(-1))对细胞的促增殖作用可分别达到137%、146%、153%。PCr(10,20 mmol·L^(-1))使MC3T3-E1细胞内ALP的活性分别增加到空白对照组的1.37和2.14倍(P<0.05,P<0.01),并且显著上调ALP蛋白表达(P<0.01,P<0.01)。PCr(5,10,20 mmol·L^(-1))还可明显上调MC3T3-E1细胞内BMP-2的蛋白表达(P<0.05,P<0.01,P<0.01),并使MC3T3-E1细胞形成的矿化结节数增加(P<0.05,P<0.01,P<0.001)。结论PCr可以促进MC3T3-E1细胞的增殖、分化与矿化,具有促成骨作用。

Objective To study the effects of phosphocreatine（ PCr） on the proliferation,differentiation and mineralization in mouse osteoblast-like MC3 T3-E1 cells. Methods MC3 T3-E1 cell cultured in vitro were treated by various concentrations of phosphocreatine（ 0,5,10,20 mmol·L^-1）. The cell proliferation was detected by MTT assay. The activity of Alkaline phosphatase（ ALP） was determined by the ALP activity assay kit. Western blot was used to evaluate the expression levels of ALP and bone morphogenetic protein-2（ BMP-2） and alizarin red staining was used to identify the mineralization of the cells. Results PCr treating had no obvious proliferative effect on MC3 T3-E1 cells in24 h（ P〉0. 05）,after 48 h,the proliferative effect of PCr（ 5,10,20 mmol·L^-1） on cells was 137%,146%,and153% compared with cells without PCr,respectively. ALP and BMP-2 expression enhanced and was ALP activity increased after PCr treatment. Moreover,PCr stimulated calcium node formation of MC3 T3-E1 cell. Conclusion PCr could promote MC3 T3-E1 cells proliferation,differentiation and mineralization and help bone formation.