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目的:研究金雀异黄酮(genistein,Ge)对缺氧培养条件下成骨细胞增殖、细胞周期、凋亡和成骨分化等的影响。方法:采用酶消化法分离培养SD大鼠颅骨成骨细胞并用三气培养箱建立缺氧模型。将细胞分为常氧对照组、缺氧对照组和缺氧加药组,其中缺氧加药组进一步分为Ge-6组、Ge-5组及Ge-4组,分别加入1×10-6,1×10-5,1×10-4mol.L-1金雀异黄酮。于缺氧处理36 h后分析各组的细胞存活率、乳酸脱氢酶漏出率、细胞凋亡率、细胞周期等,并用Real time RT-PCR法检测HIF-1α,Bcl-2及Caspase-3的基因表达情况。缺氧处理48 h后检测碱性磷酸酶活性、钙化结节数量和面积等成骨分化指标。结果:与缺氧对照组比较,金雀异黄酮可显著提高细胞存活率,降低乳酸脱氢酶漏出率,减少细胞凋亡并提高G1期细胞百分比,提高HIF-1α和Bcl-2 mRNA的表达水平,抑制Caspase-3 mRNA的表达水平,对碱性磷酸酶活性和钙化结节数量与面积等也有显著提高作用,并都呈现出剂量依赖性特点。结论:金雀异黄酮对成骨细胞缺氧损伤具有明显保护作用。
Objective: To study the effects of genistein (Ge) on proliferation, cell cycle, apoptosis and osteogenic differentiation of osteoblasts under hypoxic conditions. Methods: SD rat calvarial osteoblasts were isolated and cultured by enzymatic digestion method and hypoxia model was established by using three-gas incubator. The cells were divided into normoxia control group, hypoxia control group and hypoxia addition group. The hypoxia addition group was further divided into Ge-6 group, Ge-5 group and Ge-4 group, 6,1 × 10-5, 1 × 10-4mol.L-1 genistein. The cell viability, lactate dehydrogenase leakage rate, apoptosis rate and cell cycle of each group were analyzed after hypoxia for 36 h. HIF-1α, Bcl-2 and Caspase-3 were detected by Real time RT- Of the gene expression. After 48 h of hypoxia, the alkaline phosphatase activity, number of calcified nodules and area of osteogenic differentiation were measured. Results: Compared with hypoxia control group, genistein significantly increased cell viability, decreased lactate dehydrogenase leakage rate, decreased apoptosis and increased the percentage of cells in G1 phase and increased the expression of HIF-1α and Bcl-2 mRNA Level, inhibit Caspase-3 mRNA expression, alkaline phosphatase activity and calcified nodules number and area also increased significantly, and showed a dose-dependent characteristics. Conclusion: Genistein can significantly protect osteoblasts against hypoxia.