蛇床子素对新生大鼠成骨细胞增殖的影响及相关机制研究
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作者Author单位AddressE-Mail
李玲慧 LI Ling-hui 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203
上海市中医药研究院骨伤科研究所, 上海 201203
Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China  
丁道芳 DING Dao-fang 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203
上海市中医药研究院骨伤科研究所, 上海 201203
Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China  
杜国庆 DU Guo-qing 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203
上海市中医药研究院骨伤科研究所, 上海 201203
Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China  
龚浩 GONG Hao 中国中医科学院望京医院, 北京 100102  
王辉昊 WANG Hui-hao 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203
上海市中医药研究院骨伤科研究所, 上海 201203
Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China  
詹红生 ZHAN Hong-sheng 上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China zhanhongsheng2010@163.com 
期刊信息:《中国骨伤》2013年,第26卷,第5期,第419-422页
DOI:10.3969/j.issn.1003-0034.2013.05.017
基金项目:国家自然基金项目(编号:81073114,81001527,81102603,81173621);“中医骨伤科学”国家重点学科(编号:100508);上海领军人才项目(编号:041);上海高校“中医脊柱病损研究”创新团队建设项目(编号:2009-26);上海市科委重点项目(编号:09dZ1973800,08411950300,11DZ1973400,11XD1404600)
中文摘要:

目的: 探讨蛇床子素对体外培养的大鼠成骨细胞增殖的影响及机制。

方法: 新生SD大鼠10只, 脱颈处死后取出颅盖骨, 剪成约1 mm×1 mm的碎片, 采用胰酶预消化15 min,继以0.1 %Ⅰ型胶原酶37℃消化2次, 每次1 h,所得细胞混匀后置于5% CO2培养箱中37℃恒温培养。细胞培养48 h、28 d后行ALP染色和矿化结节染色以进行成骨性鉴定。细胞随机分为5组, 分别加入终浓度为100、50、25、12.5、0 μmol/L的蛇床子素进行干预。24、48、72 h后采用CCK-8法检测细胞增殖率, 并通过Western Blot方法检测加药1周后PCNA、β-catenin蛋白表达量。

结果: 镜下观察细胞形态不规则, 具有典型的成骨细胞形态;ALP染色及矿化结节染色均为阳性。CCK-8检测发现100 μmol/L的蛇床子素干预24 h后即可抑制成骨细胞生长, 50 μmol/L和25 μmol/L的蛇床子素于加药后48 h开始抑制成骨细胞生长, 12.5 μmol/L蛇床子素对成骨细胞的增殖无显着影响。Western Blot检测结果显示PCNA及β-catenin随蛇床子素浓度增加而表达下调。

结论: 100、50、25 μmol/L各个浓度的蛇床子素可抑制成骨细胞增殖, 12.5 μmol/L蛇床子素对细胞的增殖特性无显着影响。蛇床子素抑制成骨细胞增殖的作用可能是通过干扰Wnt/β-catenin信号通路而实现的。
【关键词】蛇床子  成骨细胞  大鼠  细胞增殖
 
Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research
ABSTRACT  

Objective: To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism.

Methods: Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm×1 mm size. After digested by trypsin for 15 min,the cranium were digested by type Ⅰcollagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5%CO2 under the condition of 37℃。 To identify the cells,ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100,50,25,12.5,0 μmol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and β-catenin protein were detected through the method of Western Blot after one week.

Results: The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 μmol/L inhibited the proliferation of osteoblast after 24 h,while the osthole with final concentrations of 50 μmol/L and 25 μmol/L displayed the inhibition effect after 48 h. The osthole of 12.5 μmol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and β-catenin protein in a dose-dependent manner.

Conclusion: The osthole with final concentrations of 100,50,25 μmol/L inhibited the proliferation of osteoblast (P<0.05). The osthole with final concentrations of 12.5 μmol/L had no obvious influence on the proliferation of osteoblast(P>0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/β-catenin signaling in osteoblast.
KEY WORDS  Fructus cnidii  Osteoblasts  Rats  Cell proliferation
 
引用本文,请按以下格式著录参考文献:
中文格式:李玲慧,丁道芳,杜国庆,龚浩,王辉昊,詹红生.蛇床子素对新生大鼠成骨细胞增殖的影响及相关机制研究[J].中国骨伤,2013,26(5):419~422
英文格式:LI Ling-hui,DING Dao-fang,DU Guo-qing,GONG Hao,WANG Hui-hao,ZHAN Hong-sheng.Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research[J].zhongguo gu shang / China J Orthop Trauma ,2013,26(5):419~422
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