右归饮对类固醇激素性股骨头坏死大鼠成骨-破骨体外共育体系中破骨细胞分化的影响

Effect of Youguiyin Decoction on osteoclast differentiation in osteoclast and osteoblast co-culture in vitro of steroid induced avascular necrosis of femoral head

目的探讨右归饮对激素性股骨头坏死(SINFH)大鼠成骨-破骨体外共育体系中破骨细胞(osteoclast,OC)生长分化的影响。方法雄性SD大鼠70只,体重(100±20)g,用随机数字表法取30只用醋酸泼尼松龙49 mg/kg·d肌内注射造模,1周后随机抽取2只造模大鼠进行组织病理观察确定造模成功。40只正常大鼠随机分为高、中、低右归饮给药组及蒸馏水组。从造模大鼠股骨、胫骨分离培养成骨细胞(osteoblast,OB)和OC诱导5 d,将OB以1×105/mL密度接种于成骨-破骨细胞共育体系中,分别以蒸馏水血清(对照组)、低(0.5 mL/100 g)、中(1.0 mL/100 g)、高(2.0 mL/100 g)浓度右归饮含药血清作用于OB-OC共育体系。用抗酒石酸酸性磷酸酶(TRAP)鉴定OC和阳性多核细胞计数,用RT-PCR方法测定RANKL mRNA表达水平变化。结果与蒸馏水组比较,高、中浓度右归饮组TRAP染色阳性破骨细胞数量均显著减少(P<0.01);RANKL基因表达水平明显降低(P<0.01),高浓度右归饮组与低、中浓度组比较RANKL基因表达水平显著降低(P<0.01、P<0.05)。结论一定浓度的右归饮对SINFH大鼠体外成骨-破骨共育体系破骨细胞形成、分化有抑制作用,以高浓度右归饮作用最强。

Objective To explore the effect of Youguiyin （YGY） decoction on osteoclast growth and differen- tiation in osteoblast-osteoclast co-culture in vitro system of steroid induced necrosis of femoral head （SINFH）. Methods 30 of 70 SD male rats （100± 20 g） were randomly selected to establish the SINFH model by steroid injection at 49 mg/kg per day. 2 rats were randomly selected to confirm the model establishment by histologically analysis. 40 nor- mal rats were randomly divided into four groups as YGY high dose group, YGY middle dose group, YGY low dose group and distilled water group. Osteoblasts and osteoclasts were isolated from the femur and tibia of model rats and were in-duced for 5 days. 1 ~ 105/mL osteoblasts were incubated into osteoblast-osteoclast co-culture system and were separately treated with distilled water （control group）, low dose YGY group （0. 5 mL/100 g）, middle dose YGY group （ I. 0 mL/100 g）, and high dose YGY group （2.0 mL/100 g）. TRAP staining was conducted to identify the osteoclast and count the multinuclear cells, and RT-PCR was conducted to test the RANKL mRNA expression level. Results Compared to the control group, the TRAP positive osteoclasts were decreased significantly in high dose and middle dose groups of YGY （P 〈 0. 01 ） , and the RANKL gene expression level was significantly decreased in all treated groups （ P 〈 0.01, P 〈 0. 05 ）. Conclusion Certain concentration of YGY containing serum can inhibit osteoclast formation and differentiation in osteoblast-osteoclast co-culture in vitro system of SINFH rats. YGY high dose group has the most significant effect among others.