糖皮质激素对大鼠激素性坏死股骨头内主要成骨调控基因表达的影响
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摘要
目的通过糖皮质激素诱导Wistar大鼠股骨头坏死模型,研究调节成骨细胞分化和成熟关键基因Runx2(runt-related transcription factor-2)、Osterix及AJ18在不同时期激素性坏死股骨头组织中的表达及蛋白质合成的变化情况,分析其在股骨头坏死发病、进展过程中的作用,为研究激素性股骨头坏死的发病机制提供新的理论基础和实验依据。
方法按20mg/kg剂量交替经大鼠双侧臀大肌注射地塞米松,每周一次；注射地塞米松后放在实验动物跑台上每周两次训练,共8周,诱导大鼠激素性股骨头坏死动物模型。HE染色光学显微镜观察骨组织、骨髓组织、骨小梁结构变化,分析每高倍视野单位面积下空骨陷窝率、单位视野面积内骨小梁面积比、脂肪细胞直径,确定造模成功。按时段提取大鼠股骨头内总RNA及总蛋白,采用实时荧光定量PCR方法从mRNA表达水平检测激素性坏死股骨头组织中Runx2、Osterix、AJ18及骨钙素的表达情况,并用Western blot方法检测各标本Runx2、Osterix、AJ18及骨钙素蛋白表达,分析糖皮质激素对其表达的影响及其在激素性股骨头坏死发病、进展过程中的作用。
结果实验第8周取股骨头标本行病理学检查,每高倍视野单位面积下空骨陷窝率(12.36%)、单位视野内骨小梁面积(56.54%)、脂肪细胞最大直径(24.52μm)均与正常组差异有统计学意义(p<0.5),造模成功。实验8、10、12周时,实验组大鼠股骨头内Runx2、Osterix及OC的基因表达、蛋白合成较对照组减少,mRNA的表达量分别为正常组的0.9621、0.3259、0.0512,0.9661、0.2026、0.0194和0.2857、0.2027、0.1583,随时间推移三者的基因表达、蛋白合成呈下降趋势。实验组大鼠股骨头内AJ18的基因表达、蛋白合成较对照组增多,AJ18 mRNA的表达在实验后8、10、12周分别为正常组的3.5487、3.6303、3.7576。
结论激素性股骨头坏死组织中Runx2/Osterix、OC在mRNA和蛋白表达水平均低于正常股骨头组织,AJ18高于正常股骨头组织,糖皮质激素可能通过下调Runx2/Osterix mRNA和上调AJ18 mRNA来抑制成骨细胞活性,抑制骨形成和骨修复,促进骨吸收,导致激素性骨质疏松和股骨头坏死；或者先造成骨质疏松后,骨强度下降,应力作用下骨小梁微骨折引起骨小梁超微结构的改变,继而出现骨修复和应力作用下骨改建,随时间推延,此病理过程反复发生,最终导致股骨头坏死。
Objective By glucocorticoid-induced femoral head necrosis of Wistar rats to study the regulation of osteoblast differentiation and maturation of key genes Runx2 (runt-related transcription factor-2), Osterix, and AJ18 at different times of hormonal necrosis of femoral head and the expression changes in protein synthesis, analysis the role in the femoral head necrosis during the disease progression, provide new theoretical basis and experimental evidence for the femoral head necrosis in the pathogenesis.
Methods Early rat model of femoral head necrosis was made with alternate injection of dexamethasone(20mg/kg) bilateral gluteus maximus once a week and twice-weekly training on laboratory animal treadmill after 8 weeks. HE staining to determine the success of osteonecrosis, the model is divided into 8 weeks,10 weeks and12 weeks groups,each group 10 animals.According to period, extract total RNA and total protein from the femoral head for real-time quantitative polymerase chain reaction(PCR), and Western blot were performed to detect the dynamic changes of the Runx2,Osterix,AJ18,OC genes expressions and proteins synthesis inside the femoral head of steroid-induced osteonecrosis in rats. Analyze the role of glucocorticoid on regulation of osteoblast maturate gene.
Results Femoral head specimens make pathological examination afer 8 weeks, under the unit area per high power field empty lacunae rate (12.36%), flat field of view of trabecular bone area (56.54%), fat cell diameter (24.52μm) were statistical diference compared with the normal group. At the post-modeling 8th,10th and 12th week, the expression of the Runx2,Osterix and OC genes, proteins synthesis in model group than the control group to reduce obviously, and the volume of mRNA expression were respectively equivalent to 0.9621,0.3259,0.0512 and 0.9661,0.2026,0.0194 and 0.2857、0.2027、0.1583 times of those in control group. Three expression had a downward trend with the longer time. The expression of the AJ18 mRNA, protein synthesis in model group at the post-modeling 8th,10th and 12th week extremely higher than those in control group, which the volume of mRNA expression were 3.5487,3.6303,3.7576 times of those in control group.
Conclusion Early steroid-induced femoral head necrosis in rats, by down-regulation Runx2/Osterix mRNA and up-regulation AJ18 mRNA, glucocorticoids may inhibit osteoblast activity, promote bone resorption, leading to osteoporosis and femoral head necrosis.
方法按20mg/kg剂量交替经大鼠双侧臀大肌注射地塞米松,每周一次；注射地塞米松后放在实验动物跑台上每周两次训练,共8周,诱导大鼠激素性股骨头坏死动物模型。HE染色光学显微镜观察骨组织、骨髓组织、骨小梁结构变化,分析每高倍视野单位面积下空骨陷窝率、单位视野面积内骨小梁面积比、脂肪细胞直径,确定造模成功。按时段提取大鼠股骨头内总RNA及总蛋白,采用实时荧光定量PCR方法从mRNA表达水平检测激素性坏死股骨头组织中Runx2、Osterix、AJ18及骨钙素的表达情况,并用Western blot方法检测各标本Runx2、Osterix、AJ18及骨钙素蛋白表达,分析糖皮质激素对其表达的影响及其在激素性股骨头坏死发病、进展过程中的作用。
结果实验第8周取股骨头标本行病理学检查,每高倍视野单位面积下空骨陷窝率(12.36%)、单位视野内骨小梁面积(56.54%)、脂肪细胞最大直径(24.52μm)均与正常组差异有统计学意义(p<0.5),造模成功。实验8、10、12周时,实验组大鼠股骨头内Runx2、Osterix及OC的基因表达、蛋白合成较对照组减少,mRNA的表达量分别为正常组的0.9621、0.3259、0.0512,0.9661、0.2026、0.0194和0.2857、0.2027、0.1583,随时间推移三者的基因表达、蛋白合成呈下降趋势。实验组大鼠股骨头内AJ18的基因表达、蛋白合成较对照组增多,AJ18 mRNA的表达在实验后8、10、12周分别为正常组的3.5487、3.6303、3.7576。
结论激素性股骨头坏死组织中Runx2/Osterix、OC在mRNA和蛋白表达水平均低于正常股骨头组织,AJ18高于正常股骨头组织,糖皮质激素可能通过下调Runx2/Osterix mRNA和上调AJ18 mRNA来抑制成骨细胞活性,抑制骨形成和骨修复,促进骨吸收,导致激素性骨质疏松和股骨头坏死；或者先造成骨质疏松后,骨强度下降,应力作用下骨小梁微骨折引起骨小梁超微结构的改变,继而出现骨修复和应力作用下骨改建,随时间推延,此病理过程反复发生,最终导致股骨头坏死。
Objective By glucocorticoid-induced femoral head necrosis of Wistar rats to study the regulation of osteoblast differentiation and maturation of key genes Runx2 (runt-related transcription factor-2), Osterix, and AJ18 at different times of hormonal necrosis of femoral head and the expression changes in protein synthesis, analysis the role in the femoral head necrosis during the disease progression, provide new theoretical basis and experimental evidence for the femoral head necrosis in the pathogenesis.
Methods Early rat model of femoral head necrosis was made with alternate injection of dexamethasone(20mg/kg) bilateral gluteus maximus once a week and twice-weekly training on laboratory animal treadmill after 8 weeks. HE staining to determine the success of osteonecrosis, the model is divided into 8 weeks,10 weeks and12 weeks groups,each group 10 animals.According to period, extract total RNA and total protein from the femoral head for real-time quantitative polymerase chain reaction(PCR), and Western blot were performed to detect the dynamic changes of the Runx2,Osterix,AJ18,OC genes expressions and proteins synthesis inside the femoral head of steroid-induced osteonecrosis in rats. Analyze the role of glucocorticoid on regulation of osteoblast maturate gene.
Results Femoral head specimens make pathological examination afer 8 weeks, under the unit area per high power field empty lacunae rate (12.36%), flat field of view of trabecular bone area (56.54%), fat cell diameter (24.52μm) were statistical diference compared with the normal group. At the post-modeling 8th,10th and 12th week, the expression of the Runx2,Osterix and OC genes, proteins synthesis in model group than the control group to reduce obviously, and the volume of mRNA expression were respectively equivalent to 0.9621,0.3259,0.0512 and 0.9661,0.2026,0.0194 and 0.2857、0.2027、0.1583 times of those in control group. Three expression had a downward trend with the longer time. The expression of the AJ18 mRNA, protein synthesis in model group at the post-modeling 8th,10th and 12th week extremely higher than those in control group, which the volume of mRNA expression were 3.5487,3.6303,3.7576 times of those in control group.
Conclusion Early steroid-induced femoral head necrosis in rats, by down-regulation Runx2/Osterix mRNA and up-regulation AJ18 mRNA, glucocorticoids may inhibit osteoblast activity, promote bone resorption, leading to osteoporosis and femoral head necrosis.
引文
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