不同月龄雌性SD大鼠成骨细胞内雌激素受体表达规律分析

目的:建立一种灵敏度高,特异性强的成骨细胞内雌激素受体检测方法,探讨不同月龄雌性SD大鼠成骨细胞内雌激素受体表达规律,以此拟描述不同年龄人类女性个体成骨细胞内雌激素受体表达的生理曲线.方法:以不同月龄雌性SD大鼠为标本,二次酶消化法收集成骨细胞并培养,倒置相差显微镜观察成骨细胞形态,钙钴法碱性磷酸酶染色、上清液碱性磷酸酶(ALP)及骨钙素(OCN)检测对成骨细胞进行鉴定,应用流式细胞仪对不同月龄雌性SD大鼠成骨细胞内雌激素受体进行检测, 根据每个成骨细胞被激发后标记-FITC的雌激素受体(ER)抗体荧光强度来表达细胞内ER的含量.结果:二次酶消化法获得的成骨细胞,形态呈多边形,碱性磷酸酶染色阳性细胞染色率80%,不同培养时间的成骨细胞和成纤维细胞上清液碱性磷酸酶和骨钙素含量相比较成骨组明显高于成纤维组,统计学检验有显著意义(p<0.01),流式细胞仪检测结果显示: 1、2月龄组ER荧光强度低表达,3月组ER荧光强度虽然呈低表达但已经显示出上升趋势,4月组以后ER荧光强度表达逐渐升高,至8、9、10月ER荧光强度表达至高峰,11月组之后各组ER表达荧光强度呈下降趋势.结论:二次酶消化法可获得较多的成骨细胞,符合成骨细胞的生物学特性,可作为成骨细胞相关研究的细胞来源.雌性SD大鼠成骨细胞内含有丰富的ER,其表达有赖于雌激素的存在.雌性SD大鼠成骨细胞内ER表达随月龄增加呈现出1、2、3月组ER低表达,4月组以后ER表达逐渐升高,至8、9、10月ER表达至高峰,11月组之后各组ER表达呈下降趋势的规律性变化.

REGULATION OF ESTROGEN RECEPTOR EXPRESSION IN DIFFERENT MONTHS SPRAGUE DAWLEG RATE OSTEOBLAST

Objective:To apply a sensitive and specific method of detecting estrogen receptor in os teoblast, study the regulation of estrogen receptor expression in different months Sprague Dawleg rats osteoblast, and then indicate estrogen receptor expression in different years women.Methods:The osteoblasts were isolated from different months Sprague Dawleg rat long bones by sequential enzyme digestion, partial osteoblasts were cultured by DMEM. Osteoblasts were detected by inverted microscope, osteocalcin and alkaline phosphatase (ALP) activity assay respectively. Estrogen receptor expression in osteoblasts of different monthes Sprague Dawleg rats were analyzed by flow cytometry technique.Results:The cells isolated by sequential enzyme digestion were osteoblasts ,which had the morphological feature biologycal characteristic of general osteoblast. The ER indicated by fluorescence label showed low expression in 1 month,2 months groups, which showed an upward trend in 3 months group, as more in 4 months group. The ER indicated by fluorescence label showed the most intensive expression in 8,9,10 month groups, and then downward trend beyond 11 month groups.Conclusions:1 There had abundant estrogen receptor in Osteoblast of female Sprague Dawleg rat , which expression depended on estrogen. 2 Estrogen receptor expression were low in 1,2,3 month groups, upward trend in 4 month , the most intensive in 8,9,10 month groups, and then descended beyond 11month groups. 3 It was a sensitive and specific method of detecting estrogen receptor in osteoblast by flow cytometry technique.