【摘要】 目的比较并探讨甲状旁腺素和阿仑膦酸钠治疗糖皮质激素性骨质疏松(GIOP)的疗效和作用机制。方法动物实验:将40只9月龄雄性SD大鼠分为对照组:0.9%氯化钠注射液皮下注射;糖皮质激素(GC)组:甲泼尼龙琥珀酸钠2.5mg/kg·d皮下注射;GC+双膦酸盐组:甲泼尼龙琥珀酸钠2.5mg/kg·d皮下注射+阿仑膦酸钠4mg/kg·d灌胃;GC+甲状旁腺激素(PTH)组:甲泼尼龙琥珀酸钠2.5mg/kg·d皮下注射+rPTH80μg/kg·d皮下注射。所有动物喂养3个月后处死。采用双能X线骨密度仪检测大鼠腰椎和股骨骨密度,用骨形态计量学方法分析大鼠股骨力学参数,收集外周血检测抗酒石酸酸性磷酸酶和骨钙素。细胞实验:将原代培养大鼠成骨细胞分为对照组;GC组:地塞米松(Dex)10-5mol/L;阿仑膦酸钠(ALN)组:ALN10-7mol/L;PTH组:人重组PTH1-3410-7mol/L;GC+ALN组:Dex10-5M+ALN10-7mol/L;GC+PTH组:Dex10-5mol/L+PTH10-7mol/L;分别采用MTT、碱性磷酸酶活性检测和茜素红染色法观察各组成骨细胞的增殖、分化和矿化情况;采用Real-timePCR检测各组成骨细胞FGF23和SOST基因表达情况。结果动物实验结果显示,GC组大鼠腰椎骨密度(0.232±0.021)和股骨骨密度(0.203±0.018)较各自对照组(0.247±0.03和0.226±0.037)明显降低(P<0.05);GC+ALN组股骨骨密度(0.224±0.03)和GC+PTH组腰椎骨密度(0.271±0.018)较GC组明显升高(P<0.05)。与对照组相比,GC组骨小梁容积、骨形成率、矿化沉积率和血BGP明显降低(P<0.05),破骨细胞表面积和血TRAP5b明显升高(P<0.05)。GC+PTH组骨小梁容积显著增加(P<0.05),GC+ALN组骨小梁容积与对照组持平。与GC组相比,GC+PTH组骨形成率和血BGP明显升高(P<0.05),GC+ALN组矿化沉积率升高(P<0.01),破骨细胞表面积和血TRAP5b降低(P<0.05)。细胞实验结果显示,GC可降低成骨细胞的增殖率和碱性磷酸酶活性,减少矿化结节形成(P<0.05),GC+PTH和GC+ALN使碱性磷酸酶活性升高至对照组水平,但PTH能著抑制矿化,ALN促进矿化。Realtime-PCR结果提示经GC干预后骨硬化蛋白(SOST)和纤维生长因子23(FGF23)表达显著升高(P<0.05),PTH抑制SOST表达(P<0.01),ALN抑制SOST和FGF23的表达(P<0.05)。结论过量GC可增加SOST和FGF23表达,与糖皮质激素性骨质疏松的骨形成降低和骨矿化受损有关。PTH和ALN可提高骨量,改善骨强度;PTH可降低SOST表达,促进骨形成;ALN可降低FGF23表达,促进骨矿化。更多还原

【Abstract】 Objective Glucocorticoid (GC) excess decreases bone mineralization and microarchitecture and lead to reduced bone strength. Both anabolic agent parathroid hormone (PTH) and anti-resorptive agent alendronate (ALN) are used to prevent and treat GC-induced bone loss, yet these bone active agents alter bone turnover by very different mechanisms. Our study was to determine how PTH and ALN alter bone quality following GC excess.Methods Nine-month-old SD male rats were treated with methylprednisolone (5 mg/kg·d) or placebo (CON). Other two groups of GC-treated rats had either PTH(80 μg/kg·d) or ALN(4 mg/kg·d) intervention. All the animals were killed after 3 months, and femur and tibia isolated for indicated detection. Bone quality and quantity measurements were measured by dual x-ray absorptiometry (DEXA) for the degree of bone mineralization, histomorphometry for bone microarchitecture, biochemistry for bone turnover. Osteoblasts isolated from newborn rats were cultured in vitro. Cells were treated with 1) CON: placebo; 2)GC: Dexamethasone 10-5mol/L; 3) ALN: Alendronate 10-7mol/L; 4)PTH: human recombinant parathyroid hormone 10-7mol/L; 5)GC+ALN: Dexamethasone 10-5mol/L+ALN 10-7mol/L; 6)GC+PTH: Dexamethasone 10-5mol/L+ PTH 10-7mol/L. Osteoblast proliferation and osteogenic activity were identified by MTT, alkaline phosphatase detection, calcium nodules were observed by Alizarin red staining, and real-time PCR were performed to monitor the expression of several key genes regulating bone formation and mineralization as FGF23 and SOST.Results Spine and Femur BMD was reduced after GC treatment, and it was restored to CON level with GC+ALN and GC+PTH at site of femur and spine separately (P<0.05). Compared to the CON, GC treatment decreased trabecular bone volume, bone formation rate, mineral apposition rate and serum BGP, but increased osteoclast surface and serum TRAP5b (P<0.05). GC+PTH increased and GC+ALN restored trabecular bone volume to the CON levels (P<0.05 compared to GC). GC+PTH increased bone formation rate and serum BGP compared to GC (P<0.05), while GC+ALN increased mineral apposition rate (P<0.01) and reduced osteoclast surface and serum TRAP5b compared to GC (P<0.05). As to osteoblasts experiments, GC treatment decrease MTT and AKP activity, reduced calcium nodules significantly (P<0.05 compared to CON), however, GC+PTH and GC+ALN promoted osteoblast MTT and AKP to the CON levels, but PTH significantly inhibited the calcium nodules, whereas ALN increased it. Realtime-PCR revealed that SOST and FGF23 were up-regulated by GC treatment (P<0.05 compared to CON), but SOST gene was down-regulated after GC+PTH treatment (P<0.01 compared to GC), while SOST and FGF23 genes were both down-regulated by GC+ALN treatment (P<0.05 compared to GC). Conclusion GC excess increased expression of gene that inhibit osteoblast activity and gene that inhibit mineralization and which were associated with reduced bone formation and bone volume. The addition of both PTH and ALN improved bone mass and bone strength during concurrent GC treatment, with PTH lowering expression of SOST and increasing bone formation; while ALN lowered the expression of SOST and FGF23 and reversed the deterioration of bone mineralization induced by GC excess.更多还原