葛根素对大鼠成骨细胞增殖和分化的影响*
王久亮1，郑忠志2，李灵芝3，张永亮3，龚海英3
Effects of puerarin on the proliferation and differentiation of osteoblasts in rats*
AbstractAIM：It has been found in the previous studies that puerarin could inhibit the bone absorption of osteoclasts and promote bone growth in vitro. In this study, the effects of puerarin on the proliferation and differentiation of osteoblasts in rat were verified and its effect on bone formation was evaluated.
METHODS：The experiment was conducted in the Drug Screening Laboratory, Department of Medicinal Chemistry, Medical Collage of Chinese People's Armed Police Forces from March 2003 to March 2004. ①Rats (Experimental Animal Center, Chinese Academy of Military Medical Sciences) were divided into 5 groups: Estrone (Zhejiang Xianju Pharmaceutical Factory) positive control group, blank control group (only with equal quantity of cells, without any drug), and 0.01, 0.1, 1 μmol/L puerarin groups (National Institute for the Control of Pharmaceutical and Biological Products). The osteoblasts were isolated from the calvaria of neonatal SD rats within 24 hours and cultured. The cells of 0.9×107 /L were seeded in the 24-well culture plates with 12 wells in each group. After the cells were cultured for 1, 3 and 5 days, the cell number of 3 wells in every group was respectively counted and the growth curve of the cells was drawn. ②The absorption was detected by MTT method at 1, 3 and 5 days, and the rates of average proliferation were calculated. The activities of alkaline phosphatase (ALP) in and out of cells were detected by PNPP approach.
RESULTS:①Compared with control group, the proliferation of cells treated with 0.1 and 1 μmol/L puerarin or estrone was accelerated significantly after cultured for 3 or 5 days (P < 0.01). ②After the cells were treated with 1 μmol/L puerarin for 1, 3 and 5 days, the activities of ALP in and out of cells were increased significantly compared with control group (P < 0.01). After the osteoblasts were treated with 0.1 μmol/L puerarin for 3 and 5 days, the activities of ALP was increased significantly compared with control group (P < 0.05, P < 0.01). After the cells were treated with 0.01 μmol/L puerarin for 5 days, the activities of ALP out of cells were increased significantly compared with control group (P < 0.01).
CONCLUSION:Puerarin promotes osteoblast to differentiate by influencing the activity of ALP and promotes bone formation by the meditation of estrogen receptor.
Wang JL, Zheng ZZ, Li LZ, Zhang YL, Gong HY.Effects of puerarin on the proliferation and differentiation of osteoblasts in rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2007;11(36):7138-7141(China)
[www.zglckf.com/zglckf/ejournal/upfiles/07-36/36k-7138(ps).pdf]
1Department of Occupational Disease, First Hospital of Inner Mongolia Medical College, Hohhot 010058, Nei Monggol Autonomous Region, China; 2Division of Medical Affairs, Dalian Hospital of Liaoning Headquarters of Chinese People's Armed Police Forces, Dalian 116013, Liaoning Province, China; 3Department of Medicinal Chemistry, Medical Collage of Chinese People's Armed Police Forces, Tianjin 300162, China
Wang Jiu-liang, Associate chief physician, Department of Occupational Disease, First Hospital of Inner Mongolia Medical College, Hohhot 010058, Nei Monggol Autonomous Region, China
Correspondence to: Li Ling-zhi, Doctor, Professor, Department of Medicinal Chemistry, Medical Collage of Chinese People's Armed Police Forces, Tianjin 300162, China
llzhx@tom.com
Supported by: the Natural Science Foundation of Tianjin, No. 033609811*
Received: 2007-02-05 Accepted: 2007-07-04
摘要
目的：课题以往的研究已证实，葛根素在体外能够抑制破骨细胞的骨吸收活性，促进骨生长，实验将进一步验证葛根素对大鼠成骨细胞增殖和分化的影响，并评价其对骨形成的作用。
方法：实验于2003－03/2004－03在武装警察部队医学院药物化学教研室药物活性筛选室进行。①实验材料：葛根素（Puerarin）购自中国药品生物制品检定所，雌酚酮购自浙江仙居制药厂。大鼠由解放军军事医学科学院实验动物中心提供。②实验方法：实验分5组：雌酚酮阳性对照组、空白对照组（不加药，只加等量细胞悬液和培养基）、0.01，0.1，1 μmol/L葛根素组。由新生24 h SD大鼠颅盖骨分离培养成骨细胞，调整成骨活细胞密度为0.9×107 L－1, 接种于24孔培养板，每组12个复孔，分别于第1，3，5 天每组取3孔计数，绘制细胞生长曲线。③实验评估：分别于第1，3，5 天采用四唑盐法测定吸光度，计算平均增殖率，采用硝基苯基质动力学法测定各组细胞内外碱性磷酸酶活性。
结果：①与空白对照组相比，0.1, 1 μmol/L葛根素干预组与雌酚酮阳性对照组第3，5 天细胞增殖速度均加快，差异有显著性意义（P < 0.01）。②经1 μmol/L葛根素处理的第1，3，5天，细胞内、外碱性磷酸酶活性均明显高于空白对照组（P < 0.01）。经0.1 μmol/L葛根素处理第3，5天，细胞内、外碱性磷酸酶活性明显高于空白对照组（P < 0.05，P < 0.01）。经0.01 μmol/L葛根素处理后第5天，细胞外碱性磷酸酶活性明显高于空白对照组（P < 0.01）。
结论：葛根素对骨形成的作用：①通过影响碱磷酸酶活性，而促进成骨细胞分化。②通过雌激素受体介导促进成骨细胞的骨形成效应。
关键词：葛根素；成骨细胞；培养；增殖；分化
王久亮，郑忠志，李灵芝，张永亮，龚海英．葛根素对大鼠成骨细胞增殖和分化的影响[J].中国组织工程研究与临床康复，2007，11(36):7138-7141 [www.zglckf.com/zglckf/ejournal/upfiles/07-36/36k-7138(ps).pdf]
1 内蒙古医学院第一附属医院职业病科，内蒙古自治区呼和浩特市 010058； 2武装警警部队辽宁总队大连医院医务处，辽宁省大连市 116013； 3武装警警部队医学院药物化学教研室，天津市 300162
王久亮，男，1962年生，内蒙古自治区呼和浩特市人，汉族，1983年内蒙古医学院毕业，副主任医师，主要从事临床康复研究。
通讯作者：李灵芝，博士，教授，武装警察部队医学院药物化学教研室，天津市
300162
llzhx@tom.com
天津市自然科学基金课题: 骨导向抗骨质疏松药物研究（033609811）*
中图分类号:R329
文献标识码:A
文章编号:1673-8225
(2007)36-07138-04
收稿日期：2007-02-05
修回日期：2007-07-04
(07-50-2-820/GW·A)
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