金雀异黄酮对体外培养成骨细胞缺氧损伤的保护作用

Protective effect of genistein on hypoxic injuries of osteoblasts cultivated in vitro

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 investigate the effect of genistein on osteoblast proliferation, cellular cycle, apoptosis and differentiation of osteoblasts cultivated under hypoxia conditions. Method ： Rat osteoblasts were isolated from calvarias by enzyme digestion and a hypoxic model was established by in a triple-gas incubator. Rat osteoblasts were grouped into the normoxic control group, the hypoxia control group and the hypoxia administration group which was subdivided into Ge-6 group, Ge-5 group and Ge-4 group, to which genistein was administered at doses of 1 × 10-6, 1 × 10-5, 1 × 10-4 mol · L-1. The cell survival rate, lactic dehydrogenase leakage rate, apoptosis and differentiation of osteoblasts were observed for each group at 3 h after hypoxia, and the gene expression of HIF-la, Bcl-2, Caspase-3 was detected by Real time RT-PCR. Forty-eight hours after hypoxia, osteogenic differentiation markers including alkaline phosphatase activity and nodules were detected. Result： Compared with the hypoxia control group, the hypoxia administration group displays a significant increase in the survival rate and a decreased in LDH leakage rate, apoptosis rate and percentage of S ＋ G2 phases. Besides, the mRNA level of HIF-1α and Bcl-2 were enhanced, the mRNA level of Caspase-3 was inhibited. Conclusion： Genistein has an effect on protecting osteoblasts from hypoxia.