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[1]孔西建,翟遠(yuǎn)坤,劉玉珂,等.補(bǔ)骨脂黃酮對(duì)乳鼠顱骨成骨細(xì)胞增殖和礦化成熟的影響[J].中醫(yī)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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補(bǔ)骨脂黃酮對(duì)乳鼠顱骨成骨細(xì)胞增殖和礦化成熟的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期數(shù):
2013年09期
頁(yè)碼:
10-15
欄目:
基礎(chǔ)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; 翟遠(yuǎn)坤; 劉玉珂; 葉進(jìn); 吳丹; 毛春煥
河南省洛陽(yáng)正骨醫(yī)院,河南 洛陽(yáng) 471002
Author(s):
Kong Xijian*; Zhai Yuankun; Liu Yuke; Ye Jin; Wu Dan; Mao Chunhuan.*
Luoyang Orthopedic-Traumatological Hospital,Luoyang 471002,Henan,China
關(guān)鍵詞:
補(bǔ)骨脂 黃酮 成骨細(xì)胞 礦化 動(dòng)物實(shí)驗(yàn)
Keywords:
Psoralea corylifolia;  Flavone;  Osteoblasts;  Mineralization;  Animal experimentation
摘要:
目的:探討補(bǔ)骨脂黃酮對(duì)體外培養(yǎng)的成骨細(xì)胞增殖和礦化成熟的影響。方法:取新生SD大鼠顱骨,體外培養(yǎng)成骨細(xì)胞,取第1代細(xì)胞進(jìn)行以下實(shí)驗(yàn)。①將細(xì)胞分為6組,A組不進(jìn)行干預(yù),B、C、D、E、F組分別采用終濃度為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的補(bǔ)骨脂黃酮進(jìn)行干預(yù),檢測(cè)細(xì)胞增殖情況。②將成骨誘導(dǎo)培養(yǎng)的細(xì)胞分為6組,a組不進(jìn)行干預(yù),b、c、d、e、f組分別采用終濃度為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的補(bǔ)骨脂黃酮進(jìn)行干預(yù),9 d后檢測(cè)堿性磷酸酶活性,將堿性磷酸酶活性最高者確定為補(bǔ)骨脂黃酮的最佳濃度。③采用ELISA法檢測(cè)a組和堿性磷酸酶活性最高組成骨誘導(dǎo)培養(yǎng)3 d、6 d、9 d、12 d和15 d時(shí)培養(yǎng)液中骨鈣素、骨形態(tài)發(fā)生蛋白-2、骨橋蛋白和I型膠原蛋白的含量。④成骨誘導(dǎo)第12天,采用茜素紅染色法檢測(cè)a組和堿性磷酸酶活性最高組鈣化結(jié)節(jié)的形成情況。⑤采用PCR法測(cè)定a組和堿性磷酸酶活性最高組成骨培養(yǎng)開(kāi)始后72 h內(nèi)堿性成纖維細(xì)胞生長(zhǎng)因子mRNA、胰島素樣生長(zhǎng)因子-1mRNA、轉(zhuǎn)錄因子Runx-2 mRNA和Osterix mRNA的表達(dá)水平。結(jié)果:①細(xì)胞增殖測(cè)定結(jié)果。6組吸光度值比較,差異無(wú)統(tǒng)計(jì)學(xué)意義[(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堿性磷酸酶活性檢測(cè)結(jié)果。6組吸光度值比較,差異有統(tǒng)計(jì)學(xué)意義[(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組吸光度值高于b組(P=0.018),低于其余4組(P=0.000,P=0.003,P=0.016,P=0.043),c組高于b、d、e、f組(P=0.000,P=0.034,P=0.013,P=0.001)。③骨相關(guān)蛋白檢測(cè)結(jié)果。除3 d外(P=0.264),c組骨鈣素分泌量在6 d、9 d、12 d和15 d均大于a組(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組骨形態(tài)發(fā)生蛋白-2分泌量在6 d、9 d和12 d均大于a組(P=0.023,P=0.001,P=0.002); 除3 d外(P=0.312),c組骨橋蛋白分泌量在6 d、9 d、12 d和15 d均大于a組(P=0.022,P=0.008,P=0.001,P=0.038); 除15 d外(P=0.785),c組Ⅰ型膠原蛋白分泌量在3 d、6 d、9 d和12 d均大于a組(P=0.042,P=0.002,P=0.003,P=0.037)。④鈣化結(jié)節(jié)形成情況。成骨誘導(dǎo)培養(yǎng)12 d后,c組的鈣化結(jié)節(jié)明顯多于a組。⑤成骨相關(guān)基因表達(dá)情況。除72 h外(P=0.255),c組堿性成纖維細(xì)胞生長(zhǎng)因子mRNA表達(dá)水平在6 h、12 h、24 h、36 h和48 h均高于a組(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組胰島素樣生長(zhǎng)因子-1 mRNA表達(dá)水平在12 h、24 h、36 h和48 h均高于a組(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組Runx-2 mRNA表達(dá)水平在12 h、24 h、36 h和48 h均高于c組(P=0.001,P=0.004,P=0.003,P=0.023); 除72 h外(P=0.271),c組Osterix mRNA表達(dá)水平在6 h、12 h、24 h、36 h和48 h均高于a組(P=0.024,P=0.001,P=0.000,P=0.000,P=0.021)。結(jié)論:補(bǔ)骨脂黃酮對(duì)體外培養(yǎng)成骨細(xì)胞的增殖并無(wú)明顯影響,但可以促進(jìn)體外培養(yǎng)的成骨細(xì)胞礦化成熟,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----------------------------------------------- 基金項(xiàng)目:國(guó)家自然科學(xué)基金資助項(xiàng)目(81073037,3071043) 通訊作者:孔西建 E-mail:[email protected] 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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備注/Memo

備注/Memo:
2012-12-05收稿 2013-04-08修回
基金項(xiàng)目:國(guó)家自然科學(xué)基金資助項(xiàng)目(81073037,3071043)
通訊作者:孔西建 E-mail:[email protected]
更新日期/Last Update: 2013-09-30