中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
10期
1976-1979
,共4页
欧阳俊%廖二元%罗湘杭%邵挥戈%周后德
歐暘俊%廖二元%囉湘杭%邵揮戈%週後德
구양준%료이원%라상항%소휘과%주후덕
雌激素%孕激素%护骨素%成骨细胞
雌激素%孕激素%護骨素%成骨細胞
자격소%잉격소%호골소%성골세포
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17β-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±014),(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.
揹景:護骨素是一種由成骨細胞錶達的蛋白,可抑製破骨細胞分化和活化.目的:觀察比較17β-雌二醇與孕激素對人成骨細胞護骨素基因錶達的調控作用.設計:對比觀察.單位:中南大學湘雅二醫院代謝內分泌研究所.材料:Ⅳ型膠原酶(Sigma公司),胎牛血清(Gibico公司),MEM(Sigma公司),骨鈣素放射免疫法測定試劑盒(Dia2Sorin公司).方法:實驗于2003-01/2006-03在中南大學湘雅二醫院代謝內分泌研究所完成.採用正常人髂前上棘鬆質骨進行成骨細胞的提取和培養,人成骨細胞用17β-雌二醇與孕酮榦預,採用Northern雜交檢測護骨素mRNA錶達及酶聯免疫吸附法檢測護骨素蛋白質錶達.主要觀察指標:①人成骨細胞鑒定.②Northern雜交分析17β-雌二醇、孕酮對人成骨細胞護骨素mRNA錶達的影響.③酶聯免疫吸附分析17β-雌二醇、孕酮對人成骨細胞護骨素蛋白質分泌的影響.結果:①人成骨細胞鑒定:人成骨細胞分泌的堿性燐痠酶活性為(74.3±4.7)U/g;培養上清液中骨鈣素濃度為(3.84±0.39)μg/L.錶明分離培養的人成骨細胞具有成骨細胞的特性.②Northern雜交分析17β-雌二醇、孕酮對人成骨細胞護骨素mRNA錶達的影響:人成骨細胞對照組護骨素mRNA錶達條帶弱,與對照組相比,1×10-10,1×10-9,1×10-8 mol/L17β-雌二醇榦預組護骨素mRNA錶達條帶逐漸增彊;與0 h相比,榦預12,24及48 h護骨素mRNA錶達逐漸增彊;孕酮對人成骨細胞護骨素mRNA錶達無影響.③酶聯免疫吸附分析17β-雌二醇、孕酮對人成骨細胞護骨素蛋白質分泌的影響:與對照組比較,1×10-10,1×10-9,1×10-8 mol/L雌二醇組護骨素蛋白質分泌增加[(0.64±0.14),(1 27±0.26),(2.34±0.35),
(3.62±0.23)μg/L,P＜0.01].與對照組相比,1×10-8 mol/L 17β-雌二醇榦預12,24,48 h,護骨素蛋白質分泌增加[(0.62±0.12)比(1.30±0.30),(3.07±014),(3.50±0.33)μg/L,P＜0.01].1×(10-10～10-8)mol/L孕酮榦預12～48 h對成骨細胞護骨素蛋白質錶達無影響(P＞0.05).結論:17β-雌二醇促進人成骨細胞護骨素基因錶達,孕酮對其無影響,錶明雌激素與孕激素對骨代謝有不同的調節機製.
배경:호골소시일충유성골세포표체적단백,가억제파골세포분화화활화.목적:관찰비교17β-자이순여잉격소대인성골세포호골소기인표체적조공작용.설계:대비관찰.단위:중남대학상아이의원대사내분비연구소.재료:Ⅳ형효원매(Sigma공사),태우혈청(Gibico공사),MEM(Sigma공사),골개소방사면역법측정시제합(Dia2Sorin공사).방법:실험우2003-01/2006-03재중남대학상아이의원대사내분비연구소완성.채용정상인가전상극송질골진행성골세포적제취화배양,인성골세포용17β-자이순여잉동간예,채용Northern잡교검측호골소mRNA표체급매련면역흡부법검측호골소단백질표체.주요관찰지표:①인성골세포감정.②Northern잡교분석17β-자이순、잉동대인성골세포호골소mRNA표체적영향.③매련면역흡부분석17β-자이순、잉동대인성골세포호골소단백질분비적영향.결과:①인성골세포감정:인성골세포분비적감성린산매활성위(74.3±4.7)U/g;배양상청액중골개소농도위(3.84±0.39)μg/L.표명분리배양적인성골세포구유성골세포적특성.②Northern잡교분석17β-자이순、잉동대인성골세포호골소mRNA표체적영향:인성골세포대조조호골소mRNA표체조대약,여대조조상비,1×10-10,1×10-9,1×10-8 mol/L17β-자이순간예조호골소mRNA표체조대축점증강;여0 h상비,간예12,24급48 h호골소mRNA표체축점증강;잉동대인성골세포호골소mRNA표체무영향.③매련면역흡부분석17β-자이순、잉동대인성골세포호골소단백질분비적영향:여대조조비교,1×10-10,1×10-9,1×10-8 mol/L자이순조호골소단백질분비증가[(0.64±0.14),(1 27±0.26),(2.34±0.35),
(3.62±0.23)μg/L,P＜0.01].여대조조상비,1×10-8 mol/L 17β-자이순간예12,24,48 h,호골소단백질분비증가[(0.62±0.12)비(1.30±0.30),(3.07±014),(3.50±0.33)μg/L,P＜0.01].1×(10-10～10-8)mol/L잉동간예12～48 h대성골세포호골소단백질표체무영향(P＞0.05).결론:17β-자이순촉진인성골세포호골소기인표체,잉동대기무영향,표명자격소여잉격소대골대사유불동적조절궤제.
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.
