雌激素在体外对大鼠成骨细胞的作用*★

课题背景：课题经审查核实被确认为上海市科学技术成果（登记号：9312007Y1897）。体内外实验已证实雌二醇可通过成骨细胞的旁分泌信号系统导致破骨细胞凋亡，从而起到抑制破骨细胞性骨吸收的作用，但其对骨形成的作用尚不统一。�

同行评价：实验探讨了雌二醇在体外对大鼠成骨细胞的影响，结果发现雌激素可促进成骨细胞增殖，提高碱性磷酸酶活性，并促使骨矿化物质在骨内沉积，这从细胞水平证实了雌激素在骨形成中的作用。

重要的概念：①原代培养的细胞纯率：指碱性磷酸酶染色的细胞与所有细胞的百分比。如纯率低于90%，说明原代培养的成骨细胞中含有其他细胞较多，将影响实验的准确性。②矿化结节：矿化结节的形成代表了成骨细胞增殖成熟并行使成骨的功能。促进骨矿化的主要细胞是成骨细胞。每个矿化结节为1个具有形成结节功能的成骨细胞经一定时期的增殖而形成。

摘要
目的:近来研究表明雌激素除抑制骨吸收外，还具有促进成骨的作用。实验拟观察雌激素在体外对大鼠成骨细胞的影响。
方法：实验于2007-03在上海复旦大学放射医学研究所骨代谢研究室完成。①实验材料：1日龄SD新生大鼠（红皮鼠）10只，清洁级，体质量约10 g 左右。雌雄不拘。②实验方法：取大鼠颅盖骨机械分离加酶消化法体外分离培养成骨细胞，取传一代细胞，以1.25×106 L-1 的密度接种于96孔细胞培养板，培养24 h 后分为两组，分别为空白对照组和10-8 mmol/L 雌激素组（加入1×10-8 mmol/L 雌二醇），继续培养72 h。③实验评估：碱性磷酸酶染色鉴定成骨细胞；四甲基偶氮唑盐法检测成骨细胞的增殖能力；PNPP法测定碱性磷酸酶活性。低倍镜下测量矿化结节面积并计算面积比，观察雌二醇对矿化能力的影响。
结果：①体外分离培养的成骨细胞碱性磷酸酶染色成阳性。原代培养的细胞纯率超过90%。②与空白对照组相比，10-8 mmol/L 雌激素组成骨细胞增殖能力及碱性磷酸酶活性显著提高（P < 0.01）；10-8 mmol/L 雌激素组单位细胞数碱性磷酸酶活性较空白对照组有上升趋势，但差异无显著性（P > 0.05）。③与空白对照组相比，10-8 mmol/L 雌激素组矿化面积及矿化面积比显著增加（P < 0.01）。
结论：雌二醇在体外可促进大鼠成骨细胞生长和增殖，提高碱性磷酸酶活性，并促进骨矿化物质在骨内沉积。
关键词：雌二醇；成骨细胞；增殖；矿化；组织构建

李蔚，孙宜萍，朱国英. 雌激素在体外对大鼠成骨细胞的作用[J].中国组织工程研究与临床康复，2008，12(11):2061-2064
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2061(ps).pdf]

1上海交通大学附属第六人民医院老年科，上海市 200233；2上海复旦大学放射医学研究所，上海市 200032

李蔚★，女，1972年生，浙江省绍兴市人，汉族，1996年上海第二医科大学毕业，硕士，主治医师，主要从事老年医学的研究。
liwei2006sh@163.
com

通讯作者：孙宜萍，教授，硕士生导师，上海交通大学附属第六人民医院老年科，上海市 200233
sunyipings@yahoo.
com.cn

上海市科委课题(034119946)*

中图分类号:R318
文献标识码:A
文章编号:1673-8225
(2008)11-02061-04

收稿日期：2007-11-24 修回日期：2008-03-08 (07-50-11-6535/WL·Y)

Abstract

AIM：Recent researches have identified that, estrogen can not only inhibit bone resorption but also promote ossification. This study was designed to investigate the effects of estrogen on rat osteoblasts (OBs) in vitro.
METHODS: The experiment was carried out at Department of Bone Metabolic, Institute of Radiation Medicine, Fudan University in March 2007.①Materials: Ten 1-day-old SD rats, irrespective of genders, cleaning grade, weighed 10 g, were used in this study.②Methods: Rat OBs were separated and cultured in vitro with the skull of mechanical separation and enzyme digestion. OBs of the first generation were transferred to 96-hole culture plate at the density of 1.25×106 L-1. After 24-hour culture, these cells were divided into blank control group and estrogen group containing 1×10-8 mmol/L estradiol (E2). Then cells were cultured for another 72 hours.③Evaluation: OBs were verified by alkaline phosphatase (ALP) staining; Proliferation ability of OBs was examined by MTT; Activity of ALP was determined by PNPP. Area and area ratio of mineralized nodus were also measured and calculated through low power lens, from which the effects of E2 on mineralization was concluded.
RESULTS: ①Positive of ALP dying, the OBs culture in vitro were regarded as positive. Cell purity of primary culture was over 90%.②Compared with the blank control group, proliferation of OBs and ALP activity of E2 group were significantly raised (P < 0.01). For ALP activity of unitary cell population, an upward tendency was showed, but there was no significant difference (P > 0.05).③Area and area ratio of mineralized nodus were significantly higher in E2 group than in blank control group (P < 0.01).
CONCLUSION: E2 can stimulate the growth and proliferation of rat OBs in vitro, raise ALP activity, and promote the deposition of bone mineralized substances in skeleton.

1Department of Gerontology, Sixth People's Hospital of Shanghai Jiao Tong University, Shanghai 200233, China; 2Institute of Radiation Medicine, Fudan University, Shanghai 200032, China

Li Wei★, Master, Attending physician, Department of Gerontology, Sixth People's Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
liwei2006sh@163.
com

Correspondence to: Sun Yi-ping, Professor, Tutor of master, Department of Gerontology, Sixth People's Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
sunyipings@yahoo.
com.cn

Supported by: a grant by Shanghai Municipal Scientific Committee, No. 034119946*

Received: 2007-11-24
Accepted: 2008-03-08