不同剂量、多疗程地塞米松对大鼠成骨细胞生物活性及Wnt信号通路的影响

Effect of different concentrations,multi-treatment course of dexamethasone on osteoblast activity and Wnt signal pathway in rats

目的研究不同剂量、多疗程地塞米松对大鼠成骨细胞生物活性及Wnt信号通路的影响,以进一步探讨Wnt信号通路在皮质激素性骨质疏松的作用。方法用改良组织法培养大鼠颅骨成骨细胞,采用倒置相差显微镜及碱性磷酸酶染色法观察成骨细胞形态,将大鼠成骨细胞分为四组,分别用含不同浓度的地塞米松(0 mol/L、10-8mol/L、10-7mol/L、10-6mol/L)的DMEM(H)培养基培养,于作用后6 h、12 h、24 h、48 h,采用RT-PCR检测Wnt信号转导途径中Wnt7b、LRP 5、LRP 6、β-catenin基因mRNA表达及Wnt通路抑制因子DKK-1基因mRNA表达;于作用后1 d、2 d、3 d、5 d、7 d,采用MTS法检测地塞米松对成骨细胞增殖的影响;于作用后21 d,采用茜素红钙结节染色及油红O染色,观察地塞米松对成骨细胞成骨及成脂活性的影响。结果与地塞米松0 mmol/L组对比,在浓度10-6mmol/L、10-7mol/L的地塞米松作用下,成骨细胞的成脂活性增强,胞浆中出现大量脂滴,成骨细胞的增殖、骨形成活性明显受到抑制;RT-PCR结果显示地塞米松10-6mmol/L、10-7mol/L组Wnt7b(32.01±0.01、31.71±0.04)、LRP 5(23.28±0.12、23.31±0.10)、LRP 6(23.03±0.18、22.31±0.54)及β-catenin基因mRNA的表达量(30.78±0.42、29.14±0.82)均显著低于0mmol/L组(Wnt7b:33.04±0.19,LRP5:23.99±0.36,LRP6:25.15±0.05,β-catenin:32.49±0.45)(P<0.05),抑制因子DKK-1基因mRNA(26.14±0.48、25.46±0.64)表达量显著高于0mmol/L组(22.18±0.82)(P<0.01),且地塞米松作用时间越长,上述改变越明显。结论长期、大剂量应用糖皮质激素可通过升高DKK-1的表达抑制成骨细胞的Wnt信号通路,从而抑制成骨细胞增殖,促进成骨细胞脂肪化,进而抑制成骨细胞的骨形成活性,这可能在皮质激素性骨质疏松中发挥重要的作用。

Objective To investigate the effect of different concentrations, multi-treatment course of dexamethasone on rat osteoblast activity and Wnt signal pathway, in order to discuss the role of Wnt signal pathway in glucocorticoid-induced osteoporosis. Methods Rat ealvaria osteoblasts were cultured using modified tissue method. Morphology of osteoblasts was observed under phase contrast microscope after ALP staining. Rat osteoblasts were divided into 4 groups, which were cultured with DMEM （H） medium containing different concentrations of dexamethasone （0 tool/L, 10-s tool/L, 10-7 tool/L, and 10 -6tool/L）. After 6 - , 12 - , 24 - , and 48-hour culturing, WntTb, LRP 5, LRP 6, and [3-catenin mRNA expression in Wnt signal pathway and DKK-1 mRNA expression, a Wnt signal pathway inhibiting factor, were detected using RT-PCR. After 1 - , 2 - , 3 - , 5 - , and 7-day culturing, the effect of dexamethasone on osteoblast proliferation was detected using MTS method. After 21-day culturing, the effect of dexamethasone on the osteogenic and adipogenie activity of osteoblasts was detected using alizarin red staining for calcium nodules and oil red 0 staining. ResultsCompared with that in group treated with 0 mg/L dexamethasona, dipogenic activity of osteoblasts increased in 10-6 mmol/L and 10-7 mol/L dexamethasone groups, with abundant lipid drople.ting in the cytoplasm. While the proliferation and osteogenic activity of 0blasts was obviously inhibited. The result of RT-PCR showed that the expression of Wnt7b （32.01 ±0.01, 31.71 ±0.04）, 0.12, 23.31 ±0.10）, LRP6 （23.03 ±0.18, 22.31 ± 0. 54） and 13-eatenin mRNA （30.78 ± 0. 42, 29, 14 ± 0, pmot/L and d eaettasgne groups were significantly lower than those in 0 mmol/L dexarnethttont grotl （ WoFT .33：0-4 0 i LRP5 ： 23.99 ± 0.36 ; LRP6：25.15 ± 0. 05 ; 13 - catenin： 32.49 ±0. 45 ; P 〈 0. 05）, Whilt Dgg --- 1 115 0. 48,25.46 ± 0.64） was significantly higher than that in 0 mmol/L group （22.18 ± 0, 82! P ）, hod e,com,e aore obvious along with the elongation of the action time of dexamethasone. Conclusion Long,,e of glucocorticoid can inhibit Wnt zigzagl pathway in osteoblasts by up-regulating the expression of DKK - 1, inhibiting the proliferation of osteoblasts, promoting genesis of osteoblasts, and inhibiting the osteogenic activity of osteoblasts. This mechanism may play an important role in coid-induced osteoporosis.