摘要
目的:观察不同剂量雌激素,卵泡雌激素及黄体生成素对大鼠成骨细胞雌激素受体β表达的影响。方法:实验于2004-02/08在哈尔滨医科大学第二临床医学院科研中心完成。①取三四个月龄雌性Wistar大鼠28只用于大鼠成骨细胞体外培养。采用二次酶消化、反复贴壁法分离纯化培养大鼠成骨细胞。观察其形态、改良钙钴法碱性磷酸酶染色,培养上清碱性磷酸酶、骨钙素测定来鉴定成骨细胞。②取2代培养的成骨细胞用无血清培养基培养24h,分为10组:对照组,1×10-10,1×10-8,1×10-6mol/L17β-雌二醇组,10,100,1000U/L黄体生成素组,1×10-7,1×10-6,1×10-5g/L卵泡刺激素组。每组3个孔。对照组加入基础培养基,其他各组分别加入相应终浓度药物,培养24～48h。大鼠成骨细胞雌激素受体表达阳性细胞数检测应用免疫组织化学法测定,以细胞浆有棕黄色颗粒沉积为阳性。成骨细胞雌激素受体β表达检测应用半定量免疫印渍酶促底物染色法以及化学发光法,以A值越高,说明雌激素受体β表达越多。结果:①成骨细胞形态及特征性鉴定:成骨细胞随培养时间的延长,细胞呈指数增长,分泌碱性磷酸酶和骨钙素,碱性磷酸酶染色阳性率为90%,表现出旺盛的分泌功能。②成骨细胞雌激素受体表达阳性细胞数检测结果:不同剂量卵泡刺激素组和黄体生成素组对雌激素受体表达无明显影响;雌激素1×10-8mol/L组和1×10-6mol/L组显著高于对照组[(90.95±5.89),(95.12±6.17),(81.44±5.37)个数/100个细胞,t=2.95,P<0.05;t=3.50,P<0.01]。③成骨细胞雌激素受体β表达结果:不同剂量卵泡刺激素及黄体生成素对雌激素受体β的表达无明显影响;随雌激素浓度的增加成骨细胞雌激素受体β表达呈上升趋势。结论:雌激素能促进成骨细胞雌激素受体β的表达,且表现出剂量依赖性。而黄体生成素、卵泡刺激素对雌激素受体β表达无直接影响。
AIM: To observe the effects of estrogen, follicle estrin and luteinizing hormone at different dosage on the expression of estrin receptor β of osteoblasts in rats. METHODS: The experiment was done in Science Research Center of Second Clinical Medical College of Harbin Medical University from February to August 2004. ① Twenty-eight female Wistar rats aged three or four months were selected. The osteoblasts were isolated and cultured in vitro and pured from female Wistar rats by twice enzyme digestion and repeated strapping. Osteoblasts characteristics were identified by Gomori method staining, measuring alkaline phosphatase. Osteoblasts were detected by supernatant phosphatase and osteocalcin. ② The second generation of cells was cultured in serum-free medium for 24 hours and divided into 10 groups. Control group, 1×10-10,1×10-8,1×10-6 mol/L17β; Aquadiol group, 10,100,1 000U/L; Luteininzing hormone group, 1×10-7,1×10-6,1×10-5 g/L and follicle stimulating hormone group, with three apertures in each group. Basic medium was added into control group, and corresponding concentration drug was added into the other groups cultured for 24 to 48 hours. The number of positive cells of estrin receptor of osteoblast in rats was detected by immunohistochemistry method. It was positive with deposition of buffy granule in cell plasm. Expression of estrin receptor β of osteoblast was detected with semi-quantu immunoblotting enzyme substrate staining method and chemiluminescence method. The higher the A result was, the more the expression of estrin receptor β. RESULTS: ① Evaluation of form and character of osteoblast: Osteoblasts performed exponential growth as the time of culture long and excreting alkaline phosphatase and osteocalcin, positive rate of alkaline phosphatase staining was 90%, which expressed productive excretion function. ② Detection result of positive cells of estrin receptor: There were no significant effects on the expression of estrin receptor in follicle stimulating hormone group and luteinizing hormone group at different dosage. The results of estrin group at 1×10-8 mol/L and at 1×10-6 mol/L were significantly higher than that in control group [(90.95±5.89),(95.12±6.17),(81.44±5.37)per 100 cells,(t=2.95, P < 0.05;t=3.50, P < 0.01)]. ③ The expression results of estrin receptor β of osteoblast: There were no significant effects of follicle stimulating hormone and luteinizing hormone on the expression of estrin receptor β at different dosage. As the increase of estrin concentration, the expression of estrin receptor β of osteoblast showed increasing trend. CONCLUSION: Estrin can accelerate the expression of estrin receptor β of osteoblast and show the dosage-dependence. Luteinizing hormone and follicle stimulating hormone has no direct effect on the expression of estrin receptor β.
出处
《中国临床康复》
CAS
CSCD
北大核心
2005年第23期172-174,i004,共4页
Chinese Journal of Clinical Rehabilitation
