[1]孔西建,翟远坤,刘玉珂,等.补骨脂黄酮对乳鼠颅骨成骨细胞增殖和矿化成熟的影响[J].中医正骨,2013,25(09):10-15.
　Kong Xijian*,Zhai Yuankun,Liu Yuke,et al.Effect of bavachin on proliferation and maturation of cranioaural osteoblast in neonatal rats[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(09):10-15.
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补骨脂黄酮对乳鼠颅骨成骨细胞增殖和矿化成熟的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年09期
页码:
10-15
栏目:
基础研究
出版日期:
2013-09-30

文章信息/Info

Title:
Effect of bavachin on proliferation and maturation of cranioaural osteoblast in neonatal rats
作者:
孔西建; 翟远坤; 刘玉珂; 叶进; 吴丹; 毛春焕
河南省洛阳正骨医院,河南 洛阳 471002
Author(s):
Kong Xijian*; Zhai Yuankun; Liu Yuke; Ye Jin; Wu Dan; Mao Chunhuan.*
Luoyang Orthopedic-Traumatological Hospital,Luoyang 471002,Henan,China
关键词:
补骨脂 黄酮 成骨细胞 矿化 动物实验
Keywords:
Psoralea corylifolia;  Flavone;  Osteoblasts;  Mineralization;  Animal experimentation
摘要:
目的:探讨补骨脂黄酮对体外培养的成骨细胞增殖和矿化成熟的影响。方法:取新生SD大鼠颅骨,体外培养成骨细胞,取第1代细胞进行以下实验。①将细胞分为6组,A组不进行干预,B、C、D、E、F组分别采用终浓度为1×10-4 mol·L-1、1×10-5 mol·L-1、1×10-6 mol·L-1、1×10-7 mol·L-1、1×10-8 mol·L-1的补骨脂黄酮进行干预,检测细胞增殖情况。②将成骨诱导培养的细胞分为6组,a组不进行干预,b、c、d、e、f组分别采用终浓度为1×10-4 mol·L-1、1×10-5 mol·L-1、1×10-6 mol·L-1、1×10-7 mol·L-1、1×10-8 mol·L-1的补骨脂黄酮进行干预,9 d后检测碱性磷酸酶活性,将碱性磷酸酶活性最高者确定为补骨脂黄酮的最佳浓度。③采用ELISA法检测a组和碱性磷酸酶活性最高组成骨诱导培养3 d、6 d、9 d、12 d和15 d时培养液中骨钙素、骨形态发生蛋白-2、骨桥蛋白和I型胶原蛋白的含量。④成骨诱导第12天,采用茜素红染色法检测a组和碱性磷酸酶活性最高组钙化结节的形成情况。⑤采用PCR法测定a组和碱性磷酸酶活性最高组成骨培养开始后72 h内碱性成纤维细胞生长因子mRNA、胰岛素样生长因子-1mRNA、转录因子Runx-2 mRNA和Osterix mRNA的表达水平。结果:①细胞增殖测定结果。6组吸光度值比较,差异无统计学意义[(0.512±0.046),(0.448±0.051),(0.528±0.043),(0.525±0.041),(0.522±0.039),(0.517±0.049),F=1.438,P=0.282]。②碱性磷酸酶活性检测结果。6组吸光度值比较,差异有统计学意义[(2.637±0.221),(2.136±0.168),(3.678±0.235),(3.153±0.201),(3.001±0.224),(2.934±0.188),F=15.442,P=0.000]; a组吸光度值高于b组(P=0.018),低于其余4组(P=0.000,P=0.003,P=0.016,P=0.043),c组高于b、d、e、f组(P=0.000,P=0.034,P=0.013,P=0.001)。③骨相关蛋白检测结果。除3 d外(P=0.264),c组骨钙素分泌量在6 d、9 d、12 d和15 d均大于a组(P=0.027,P=0.002,P=0.005,P=0.033); 除3 d和15 d外(P=0.085,P=0.132),c组骨形态发生蛋白-2分泌量在6 d、9 d和12 d均大于a组(P=0.023,P=0.001,P=0.002); 除3 d外(P=0.312),c组骨桥蛋白分泌量在6 d、9 d、12 d和15 d均大于a组(P=0.022,P=0.008,P=0.001,P=0.038); 除15 d外(P=0.785),c组Ⅰ型胶原蛋白分泌量在3 d、6 d、9 d和12 d均大于a组(P=0.042,P=0.002,P=0.003,P=0.037)。④钙化结节形成情况。成骨诱导培养12 d后,c组的钙化结节明显多于a组。⑤成骨相关基因表达情况。除72 h外(P=0.255),c组碱性成纤维细胞生长因子mRNA表达水平在6 h、12 h、24 h、36 h和48 h均高于a组(P=0.027,P=0.009,P=0.002,P=0.006,P=0.022); 除6 h和72 h外(P=0.092,P=0.114),c组胰岛素样生长因子-1 mRNA表达水平在12 h、24 h、36 h和48 h均高于a组(P=0.007,P=0.005,P=0.003,P=0.026); 除6 h和72 h外(P=0.186,P=0.359),c组Runx-2 mRNA表达水平在12 h、24 h、36 h和48 h均高于c组(P=0.001,P=0.004,P=0.003,P=0.023); 除72 h外(P=0.271),c组Osterix mRNA表达水平在6 h、12 h、24 h、36 h和48 h均高于a组(P=0.024,P=0.001,P=0.000,P=0.000,P=0.021)。结论:补骨脂黄酮对体外培养成骨细胞的增殖并无明显影响,但可以促进体外培养的成骨细胞矿化成熟,有一定的促骨形成活性。
Abstract:
Objective:To explore the effect of bavachin on proliferation and maturation of osteoblast cultured in vitro.Methods:The skulls were taken from SD neonatal rats for osteoblast culture,and the first-generation cells were chosen for the following experiments.The cells were divided into 6 groups,and cells in group A were not intervened,while cells in other groups were placed in the culture fluids respectively added with bavachin which final concentration were 1×10-4 mol/L(group B),1×10-5 mol/L(group C),1×10-6 mol/L(group D),1×10-7 mol/L(group E),1×10-8 mol/L(group F),and the cells proliferation were detected.The cells were divided into 6 groups after osteogenic induction,and cells in group a were not intervened,while cells in other groups were placed in the culture fluids----------------------------------------------- 基金项目:国家自然科学基金资助项目(81073037,3071043) 通讯作者:孔西建 E-mail:kxjian1962@126.com respectively added with bavachin which final concentration were 1×10-4 mol/L(group b),1×10-5 mol/L(group c),1×10-6 mol/L(group d),1×10-7 mol/L(group e),1×10-8 mol/L(group f).The alkaline phosphatase(ALP)activities were detected 9 days later and the optimal concentration of bavachin was determined by the highest ALP activities.The content of osteocalcin(OC),bone morphogenetic proteins-2(BMP-2),osteopontin(OPN)and type-Ⅰ collagen protein in the culture solution of group a and the group with the highest ALP activity were detected through ELISA 3,6,9,12 and 15 days after osteogenic induction respectively.The numbers of calcified nodules in group a and the group with the highest ALP activity were detected through alizarin red staining 12 days after osteogenic induction.The expression level of basic fibroblast growth factor(bFGF)mRNA,insulin-like growth factor-1(IGF-1)mRNA,Runx-2 mRNA and Osterix mRNA in group a and the group with the highest ALP activity were detected through PCR within 72 hours after the culture.Results:The cells proliferation detection showed there were no statistical differences in optical density(OD)among group A,B,C,D,E and F((0.512+/-0.046),(0.448+/-0.051),(0.528+/-0.043),(0.525+/-0.041),(0.522+/-0.039),(0.517+/-0.049),F=1.438,P=0.282).The ALP activities detection showed there were statistical differences in OD among group a,b,c,d,e and f((2.637+/-0.221),(2.136+/-0.168),(3.678+/-0.235),(3.153+/-0.201),(3.001+/-0.224),(2.934+/-0.188),F=15.442,P=0.000); The OD of group a was higher than that of group b(P=0.018)and lower than that of the other 4 groups(P=0.000,P=0.003,P=0.016,P=0.043),and the OD of group c was higher than that of group b,d,e and f(P=0.000,P=0.034,P=0.013,P=0.001).The content of OC of group c was higher than that of group a 6,9,12 and 15 days after the culture(P=0.027,P=0.002,P=0.005,P=0.033).The content of BMP-2 of group c was higher than that of group a 6,9 and 12 days after the culture(P=0.023,P=0.001,P=0.002).The content of OPN of group c was higher than that of group a 6,9,12 and 15 days after the culture(P=0.022,P=0.008,P=0.001,P=0.038).The content of type-Ⅰcollagen protein of group c was higher than that of group a 3,6,9 and 12 days after the culture(P=0.042,P=0.002,P=0.003,P=0.037).The number of calcified nodules of group c was obviously more than that of group a 12 days after the culture.The expression level of bFGF mRNA of group c was higher than that of group a 6,12,24,36 and 48 hours after the culture(P=0.027,P=0.009,P=0.002,P=0.006,P=0.022).The expression level of IGF-1 mRNA of group c was higher than that of group a 12,24,36 and 48 hours after the culture(P=0.007,P=0.005,P=0.003,P=0.026).The expression level of Runx-2 mRNA of group c was higher than that of group a 12,24,36 and 48 hours after the culture(P=0.001,P=0.004,P=0.003,P=0.023).The expression level of Osterix mRNA of group c was higher than that of group a 6,12,24,36 and 48 hours after the culture(P=0.024,P=0.001,P=0.000,P=0.000,P=0.021).Conclusion:Bavachin have no obvious effect on proliferation of osteoblasts cultured in vitro,while it can promote the mineralization and maturity of osteoblasts cultured in vitro,so it has promotive effect on bone formation.

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备注/Memo

备注/Memo:
2012-12-05收稿 2013-04-08修回
基金项目:国家自然科学基金资助项目(81073037,3071043)
通讯作者:孔西建 E-mail:kxjian1962@126.com
更新日期/Last Update: 2013-09-30