目的 研究杜仲叶提取物对大鼠成骨细胞的增殖作用及其分子机制.方法 选取新生SD大鼠的乳鼠颅骨通过消化法分离出乳鼠成骨细胞,并用碱性磷酸酶染色进行细胞鉴定；先用杜仲叶提取物分别以O mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度梯度对大鼠成骨细胞干预,2d后用MTT方法检测细胞增殖情况；用无血清无酚红的培养基饥饿大鼠成骨细胞2h后,分别以0mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度杜仲叶提取物干预大鼠成骨细胞,2h后Western blot检测ERK和AKT的活化情况.结果 碱性磷酸酶染色后的成骨细胞呈紫红色(特异性染色),MTT法结果显示杜仲叶提取物促进大鼠成骨细胞的增殖,并具有浓度依赖性；Western blot结果显示杜仲叶提取物可使ERK及AKT磷酸化水平提高,并具有浓度依赖性.结论 杜仲叶提取物通过ERK通路及AKT通路促进大鼠成骨细胞的增殖.%Objective To investigate the effect of the extraction of eucommia leaves on rat osteoblast proliferation and the molecular mechanism. Methods The osteoblasts were isolated and cultured from the calvaria of new-born SD rats. The cells were identified with alkaline phosphatase staining. Rat osteoblasts were treated with 5 different concentration gradients of leaf extraction; 0 mg/ml, 5 mg/ml, 20 mg/ml, 60 mg/ml, and 100 mg/ml. Cell proliferation was detected using MTT method 2 days later. After the addition of serum-free and phenol red-free medium for 2 h, 0 mg/ml, 5 mg/ml, 20 mg/ml, 60 mg/ml, or 100 mg/ ml leaf extraction was additioned to the medium of the osteoblast culture. Then the expression of ERK and AKT was detected using Western blotting 2 hours later. Results After alkaline phosphatase staining, osteoblasts were stained in purple ( specific staining). The results of MTT methods showed that leaf extraction promoted rat osteoblast proliferation in a dose-dependent manner. The results of Western blotting showed that leaf extraction could improve the phosphorylation levels of ERK and AKT in a dose - dependent manner. Conclusion The extraction of eucommia leaves promotes rat osteoblast proliferation through ERK and AKT pathways.
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