雌二醇对骨形态发生蛋白受体表达的影响

目的研究骨髓基质细胞向成骨细胞分化过程中,雌二醇对其骨形态发生蛋白受体(BMPR)Ⅰ A,Ⅰ B mRNA表达的影响,探讨雌二醇对成骨细胞生成的作用,试阐明绝经后高转换骨代谢始动环节的可能机制.方法SD大鼠骨髓基质细胞在生长培养基中传代培养后,用 1,25(OH)2D3和地塞米松(DEX)诱导骨髓基质细胞向成骨细胞分化, 17β-雌二醇( E2)处理培养细胞,观察E2对骨髓基质细胞分化过程中骨形态发生蛋白受体Ⅰ A、Ⅰ B mRNA、碱性磷酸酶(ALP)活性和Ⅰ型胶原合成的影响,β- actin作内对照半定量 RT- PCR分析骨形态发生蛋白受体ⅠA,Ⅰ B mRNA的表达,以α-磷酸奈酚为底物,测定细胞碱性磷酸酶的活性,Van Gieson染色法显示Ⅰ型胶原的含量. 结果E2能明显抑制骨髓基质细胞分化过程中BMPR-Ⅰ A mRNA的表达,且呈剂量依赖性,随 E2浓度增加( 0～ 1× 103 nmol/L)BMPR-Ⅰ A mRNA从 (27.0± 3.4)%降至 (17.0± 1.8)% ( t=5.2,P< 0.01)和 (9.3± 1.6)%(t=9.2,P< 0.01); BMPR-Ⅰ B mRNA随 E2浓度增加而增加,从 (2.0± 0.8)%增至 (4.8±1.5)% ( t=3.3,P< 0.05)和 (17.2± 2.2)% (t=13,P< 0.01). Northern blot结果显示在上述 E2浓度时 BMPR-Ⅰ A mRNA的表达从 4.21± 0.36降至 1.24±0.10( t=18.2,P< 0.01); BMPR-Ⅰ B mRNA的表达从 1.72± 0.11增至 3.73± 0.31(t=12.2,P< 0.01). ALP活性随 E2浓度增加而降低,从 (42.6± 2.5)U/g蛋白降至(17.9± 2.0)U/g蛋白 ( t=15,P< 0.01) ,(10.8± 1.5)U/g蛋白( t=22,P< 0.01)和 (3.6±0.7)U/g蛋白( t=30,P< 0.01);细胞Ⅰ型胶原的含量随 E2浓度增加而减少,Van Gieson染色由鲜红、淡红到黄色,红色越深,表明胶原越多. 结论E2能明显抑制骨髓基质细胞分化过程中BMPR-Ⅰ A mRNA的表达,降低 ALP的活性和Ⅰ型胶原含量,增加 BMPR-Ⅰ B mRNA的表达).

Effects of 17β- estradiol on the gene expression of bone morphogenetic protein receptor

AIM:To investigate the effects of 17β- estradiol (E2) on the gene expression of typeⅠ A and typeⅠ B bone morphogenetic protein receptor (BMPR-Ⅰ A,Ⅰ B) in rat bone marrow stromal cells exposured to the differentiation medium and to elucidate the effects of E2 on osteoblastogenesis. METHODS:Adherent bone marrow stromal cells were cultured in differentiation medium containing DEX(1× 10- 7 mol/L)and 1,25(OH)2D3 (1× 10- 9 mol/L )and different concentrations of E2.The gene expression of BMPR-Ⅰ A,Ⅰ B was quantified by semiquantitative RT- PCR and demonstrated by Northern blot. RESULTS:E2 evidently inhibited the expression of BMPR-Ⅰ A mRNA in bone marrow stromal cell.The suppression was dose dependent.When examined under various concentrations of E2 (0- 1× 10- 6 mol/L),the expression of BMPR-Ⅰ A mRNA were decreased from (27.0± 3.4)% to (22.3± 2.6)% (t=2.2,P >0.05),(17.0± 1.8)%( t=5.2,P< 0.01) and (9.3± 1.6)%( t=9.2,P< 0.01) .Increase of BMPR-Ⅰ B mRNA with increasing of E2 concentration[from (2.0± 0.8)% to (4.8± 1.5)% ,t=3.3,P< 0.05],(8.4± 1.4)%( t=7.9,P< 0.01) and (17.3± 2.2)%( t=13.1,P< 0.01) above the control. Northern blot showed the expression of BMPR-Ⅰ A mRNA decreased from 4.21± 0.36 to 1.24± 0.10( t=18.2,P< 0.01) and the expression of BMPR-Ⅰ B mRNA increased from 1.72 ± 0.11 to 3.73± 0.31( t=12.2,P< 0.01) .The activity of ALP decreased significantly at higher concentrations of E2,from (42.6± 2.5) U/g to (17.9± 2.0) U/g(t=15,P< 0.01), (10.8 ± 1.5) U/g(t=22,P< 0.01) and (3.6± 0.7) U/g( t=30,P< 0.01) . The amount of type Ⅰ collagen was suppressed by E2. CONCLUSION:E2 can suppress the gene expression of BMPR-Ⅰ A of bone marrow stromal cells and inhibited osteoblastogenesis in vitro.