不同剂量和作用时间地塞米松对大鼠骨密度和成骨细胞作用的研究

Effect of dexamethasone at different doses and courses on bone mineral density and osteoblasts in rats

目的观察地塞米松(Dex)不同剂量及不同作用时间对大鼠骨量、成骨细胞的影响。方法 120只3月龄SD雌性大鼠,随机分对照组(生理盐水)、小剂量组(地塞米松1 mg/kg)、中剂量组(地塞米松2.5 mg/kg)、高剂量组(地塞米松5 mg/kg),每组30只,2次/周肌内注射,分别干预4 w、9 w、12 w。给药前及干预后采用双能X线骨密度仪测大鼠全身骨密度(BMD),免疫组化法检测骨组织碱性磷酸酶(ALP)、I型胶原蛋白表达。从新生SD大鼠颅骨体外分离培养成骨细胞,随机分为5组:空白对照组、5×10-8mol/L Dex组、5×10-7mol/L Dex组、5×10-6mol/L Dex组、5×10-5mol/L Dex组,分别培养12h、24 h、48 h后,采用CCK8法检测成骨细胞增殖,采用RT-PCR检测成骨细胞ALP mRNA、I型胶原mRNA表达。结果 (1)干预4 w,不同剂量Dex组大鼠的骨密度与对照组无差异(P>0.05)。干预9 w,不同剂量Dex组大鼠的骨密度均低于对照组(P<0.05),高剂量组大鼠骨密度低于小剂量组(P<0.05);干预12 w,不同剂量Dex组大鼠的骨密度均低于对照组(P<0.05),且随着剂量的增加,大鼠的骨密度逐渐降低。(2)干预4 w,ALP、I型胶原蛋白表达与对照组无差异(P>0.05);干预9w,中剂量和高剂量组大鼠骨组织I型胶原蛋白表达均低于对照组(P<0.05),不同剂量组大鼠骨组织ALP蛋白表达均低于对照组(P<0.05);干预12 w,大鼠骨组织ALP、I型胶原蛋白表达均低于对照组(P<0.05);随着Dex剂量的增加,大鼠骨组织ALP及I型胶原蛋白表达逐渐降低。(3)体外分离培养成骨细胞干预12 h,5×10-6mol/L Dex和5×10-5mol/L Dex均抑制成骨细胞增殖(P<0.05),5×10-5mol/L Dex组成骨细胞的ALP、I型胶原mRNA表达减少(P<0.05);干预24 h及48 h,不同浓度Dex均明显抑制成骨细胞增殖(P<0.05),ALP、I型胶原mRNA表达明显减少(P<0.05),呈剂量和时间依赖性。结论地塞米松可能通过抑制成骨细胞的增殖及骨形成,导致SD大鼠骨密度降低。

Objective To investigate the effect of dexamethasone （Dex） on rat bone mass and osteoblasts at different dose and time.Methods A total of 120 3-month-old SD rats were randomly divided into control group （Cont, saline）, low-dose Dex group （LDG, 1 mg/kg）, medium-dose Dex group （MDG, 2.5 mg/kg） and high-dose Dex group （HDG, 5 mg/kg）, with 30 rats in each group.Dex was injected intramuscularly twice a week for 4, 9, and 12 weeks.BMD was measured using dual energy X ray absorptiometry.The protein expression of ALP and type Ⅰ collagen in bone tissue was measured with immunohistochemistry.The osteoblasts were collected fromol/L the calvaria of newborn SD rats and cultured.They were then randomly divided into 5 groups：blank group, 5×10^-8mol/L group, 5×10^ -7mol/L group, 5×10^ -6mol/L group, and 5×10 ^-5mol/L group.Cells were cultured for 12, 24, or 48 h.Cell proliferation was measured with CCK8 method.The expressions of ALP and type Ⅰ collagen mRNA were measured with RT-PCR.Results （1） After 4-week intervention, BMD in different doses of Dex groups were not different compared with control group （P〉0.05）.After 9-week treatment, BMD in different doses of Dex groups were lower than that of control group （P〈0.05）.BMD was significantly lower in HDG groups than those in LDG group （P〈0.05）.After 12-week intervention, BMD was significantly lower in Dex groups than in control group in a dose-dependent manner.（2） The expressions of ALP and type Ⅰ collagen protein were not different between Dex groups and control group in 4 weeks （P〉0.05）.The expressions of ALP and type Ⅰ collagen protein were lower in MDG and HDG groups than in control group in 9 and 12 weeks （P〈0.05）.（3） At 12 h, the proliferation of osteoblasts at the concentrations of 5 ×10^-6mol/L and 5×10 ^-5mol/L groups were inhibited （P〈0.05）.The expressions of ALP and type Ⅰ collagen mRNA in 5×10 ^-5mol/L group were lower than in blank group （P〈0.05）.At 24 h and 48 h, the proliferation and expression of ALP and type Ⅰ collagen mRNA in different concentrations of Dex groups were lower than those in blank group （P〈0.05）.Conclusion Dex inhibits osteoblast proliferation and bone formation, leading to a decrease of BMD in rats.