雌二醇与孕激素对人成骨细胞护骨素基因作用的比较

背景: 护骨素是一种由成骨细胞表达的蛋白, 可抑制破骨细胞分化和活化。目的: 观察比较 17 β- 雌二醇与孕激素对人成骨细胞护骨素基因表达的调控作用。设计: 对比观察。单位: 中南大学湘雅二医院代谢内分泌研究所。材料: Ⅳ型胶原酶( Sigma 公司), 胎牛血清(Gibico 公司), MEM(Sigma公司), 骨钙素放射免疫法测定试剂盒(Dia2Sorin 公司)。方法: 实验于 2003- 01/2006- 03 在中南大学湘雅二医院代谢内分泌研究所完成。采用正常人髂前上棘松质骨进行成骨细胞的提取和培养, 人成骨细胞用 17β- 雌二醇与孕酮干预, 采用 Northern 杂交检测护骨素mRNA 表达及酶联免疫吸附法检测护骨素蛋白质表达。主要观察指标: ①人成骨细胞鉴定。②Northern 杂交分析 17β- 雌二醇、孕酮对人成骨细胞护骨素 mRNA 表达的影响。③酶联免疫吸附分析17β- 雌二醇、孕酮对人成骨细胞护骨素蛋白质分泌的影响。结果: ①人成骨细胞鉴定: 人成骨细胞分泌的碱性磷酸酶活性为(74.3±4.7) U/g; 培养上清液中骨钙素浓度为(3.84±0.39) μg/L。表明分离培养的人成骨细胞具有成骨细胞的特性。②Northern 杂交分析 17β- 雌二醇、孕酮对人成骨细胞护骨素 mRNA 表达的影响: 人成骨细胞对照组护骨素 mRNA 表达条带弱, 与对照组相比, 1×10-10, 1×10-9, 1×10-8 mol/L17β- 雌二醇干预组护骨素 mRNA 表达条带逐渐增强; 与 0 h 相比, 干预12, 24 及 48 h 护骨素 mRNA 表达逐渐增强; 孕酮对人成骨细胞护骨素mRNA表达无影响。③酶联免疫吸附分析 17β- 雌二醇、孕酮对人成骨细胞护骨素蛋白质分泌的影响: 与对照组比较, 1×10 -10, 1×10-9, 1×10-8 mol/L雌二醇组护骨素蛋白质分泌增加[(0.64 ±0.14), (1.27±0.26), (2.34±0.35),(3.62 ±0.23) μg/ L, P < 0.01]。与对照组相比, 1×10-8 mol/L 17β- 雌二醇干预 12, 24, 48 h, 护骨素蛋白质分泌增加[(0.62±0.12)比(1.30±0.30),(3.07±0.14), (3.50±0.33) μg/L, P < 0.01]。1×(10-10～10-8) mol/L 孕酮干预 12～48 h 对成骨细胞护骨素蛋白质表达无影响(P > 0.05)。结论:17β- 雌二醇促进人成骨细胞护骨素基因表达, 孕酮对其无影响,表明雌激素与孕激素对骨代谢有不同的调节机制。

17 beta-estradiol versus progesterone in the expression of osteoprotegerin gene in human osteoblast-like cells

BACKGROUND:Estrogen/progestins replacement therapy prevents excess bone loss in postmenopausal women.Recently osteoprotegerin (OPG) has been identified in osteoblast and displayed to inhibit bone resorption.OBJECTIVE: To compare the action between 17β-estradiol (E2) and progesterone on OPG expression in cultured normal human osteoblast-like cells (hOB).DESIGN: A comparative investigation.SETTING: Institute of Metabolic Endocrinology, the Second Xiangya Hospital of Central South University.MATERIALS: α-MEM (Sigma Chemical Corp., St. Louis, MO, USA); Type Ⅳ collagenase (Sigma); Fetal bovine serum (Gibco-BRL Corp., Grand Island, NY, USA); Osteocalcin radioimmunoassay kit (DiaSorin Corp., Stillwater, MN, USA).METHODS: The experiments were carried out in the Institute of Metabolic Endocrinology, Second Xiangya Hospital of Central South University from January 2003 to March 2006. The osteoblasts were extracted from the cancelous bone of anterior superior iliac spine of normal people, then cultured. The hOB were treated with E2 and progesterone, and the expressions of OPG mRNA and OPG protein were determined by Northern blot analysis and enzyme-linked immunoabsorbent assay (ELISA) respectively.MAIN OUTCOME MEASURES: ①Characterization of human osteoblast-like cells; ②Effect of E2 and progesterone on OPG mRNA levels by Northern blot analysis; ③ Effect of E2 and progesterone on OPG protein levels in the conditioned medium by ELISA.RESULTS: ① Characterization of hOB in vitro The ALP levels in normal human osteoblasts were (74.3±4.7) U/g protein,and the detectable osteocalcin levels was (3.84±0.39) μg/L protein], which suggested that osteoblasts were the primary cell type found in our bone-derived cell cultures from donors. ② Effects of E2 and progesterone on the levels of OPG mRNA by Northern blot analysis: The OPG mRNA band was week in the control group [(12.3±3.5)%], treatment with 1 × 10-10, 1 ×10-9 1 ×10-8 mol/L E2 caused an increase in the levels of OPG mRNA. The expression of OPG mRNA in the 1×10-8 mol/L E2 group was gradually increased at 12, 24 and 48 hours. Progesterone had no influence on OPG mRNA expression. ③ Effects of E2 and progesterone on OPG protein production in conditioned medium determined with ELISA:ELISA revealed that treatment with 1 ×10-10, 1 ×10-9, 1 ×10-8 mol/L E2 induced obvious increase in the levels of OPG protein in cells media as compared with that in the control group [(1.27±0.26), (2.34±0.35), (3.62±0.23), (0.64±0.14)μg/L, P ＜ 0.01]. In the presence of 1×10-8 mol/L E2, OPG protein production in cells media at 12, 24 and 48 hours were significantly higher than that in the control group [(1.30±0.30), (3.07±0.14), (3.50±0.33), (0.62±0.12) μg/L, P ＜ 0.01]. 1 × 10-10, 1 ×10-9 1 × 10-8 mol/L progesterone had no influence on the OPG protein production after 12-24 hours (P ＞ 0.05).CONCLUSION: The different regulation of OPG production in osteoblasts by E2 and progesterone may contribute to the mechanisms by which estrogen or progestins exerts its different action on bone resorption.