雌二醇与抗坏血酸对成骨细胞的协同作用

The synergistic action of estradiol and L-ascorbic acid on the osteoblast-like cell line MG-63

目的 研究雌二醇 (17β estradiol,E2 )和抗坏血酸 (VitC)对成骨肉瘤样细胞株MG 6 3的协同作用 ,从而探讨E2 治疗骨质疏松的作用机制。 方法 利用 10 8mol/L的E2 与 10 0 μg/ml的VitC单独或共同处理成骨样细胞株MG 6 3,3 H掺入法 (3 H TdR)测定细胞的增殖 ,α 磷酸奈酚法测细胞内碱性磷酸酶 (ALP)活性的变化 ,半定量逆转录聚合酶链式反应 (RT PCR)测I型胶原 (collagenI)、基质金属蛋白酶 (matrixmetalloproteinase ,MMP) 1、MMP 2及护骨素 (osteoprotegerin ,OPG)基因表达的变化 ,明胶酶谱分析测MMP 2的活性 ,观察E2 及VitC对上述指标的影响。 结果 E2 与VitC合用较单用能使细胞内ALP活性增至对照组的 2倍 (1 0 4± 0 0 5对 1 99± 0 6 2 ,P <0 0 0 5 ) ,3 H TdR掺入量增至对照组的6倍 (4 386± 430对 2 6 5 37± 146 0 ,P <0 0 0 1) ,MMP 1表达降低为原来的 1/ 6 (P <0 0 5 ) ,OPG基因表达增至对照组的 9倍 (P <0 0 1)。明胶酶谱分析显示VitC使MMP 2分泌增加 ,而E2 使其分泌减少 ,二者合用对其无明显影响。 结论 VitC能使E2 作用加强 ,二者协同在绝经后妇女的骨质疏松治疗中发挥重要作用。

Objective To observe the synergistic action of 17β estradiol(E 2) and L ascorbic acid on the activity of alkaline phosphatase (ALP), cell proliferation and gene expression in osteosarcoma MG 63 cell line, and to study the mechanism of E 2 in treating osteoporosis. Methods MG 63 cells were treated with 10 8 mol/L E 2 with or without 100 μg/L L ascorbic acid, and then the ALP activity was determined by using α nitrophenyl phosphate as a substrate. Cell proliferation was measured by ( 3H) thymidine( 3H TdR) incorporation method. mRNA expression of type I collagen, matrix metalloproteinase 1(MMP 1), MMP 2 and osteoprotegerin(OPG) was detected by semiquantitative RT PCR method. Gelatin zymogram was used for the assay of MMP 2 activity. The synergistic action of E 2 and L ascorbic acid on MG 63 cell lines could be evaluated basing on the above indexes since they stand for separately the cell proliferation, gene expression and cell differentiation. Results The action of combined treatment with E 2 and L ascorbic acid on MG 63 cells was obviously stronger than the action using single E 2 or single L ascorbic acid. E 2 and L ascorbic acid increased the ALP activity to 200% ( P <0.005) as compared to the control. They downregulated the MMP 1 gene expression to 16 7% ( P <0 05) and upregulated the OPG gene expression to 9 folds ( P <0 01) The 3 H TdR incorporation in cooperatively treated MG 63 cells was six folds as that in the controls ( P <0 001) E 2 reduced while L ascorbic acid enhanced the MMP 2 secretion, but their cooperative use showed little effect on its secretion. Conclusions L ascorbic acid enhanced the effect of E 2 on the osteoblast like cell MG 63, showing synergistic action and their synergistic action palyed an important role in the treatment of osteoporosis.