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[1]朱旭貞,葉永玲,宋遠(yuǎn)江,等.鎂黃長石浸提液促進(jìn)人脂肪干細(xì)胞體外成骨分化的機(jī)制研究[J].中醫(yī)正骨,2013,25(05):3-8.
　ZHU Xu-zhen*,YE Yong-ling,SONG Yuan-jiang,et al.Mechanistic studies on the extracts from akermanite promoting the human adipose derived stem cell osteogenetic differentiation in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(05):3-8.
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鎂黃長石浸提液促進(jìn)人脂肪干細(xì)胞體外成骨分化的機(jī)制研究()
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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05期
頁碼:
3-8
欄目:
基礎(chǔ)研究
出版日期:
2013-05-31

文章信息/Info

Title:
Mechanistic studies on the extracts from akermanite promoting the human adipose derived stem cell osteogenetic differentiation in vitro
作者:
朱旭貞1; 葉永玲1; 宋遠(yuǎn)江2; 肖治剛3; 戴建英4
1.浙江省杭州市中醫(yī)院,浙江 杭州 310007; 2.浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院, 浙江 杭州 310009; 3.上海交通大學(xué)醫(yī)學(xué)院附屬第九人民醫(yī)院,上海 200011; 4.杭州市啟迪生物有限公司,浙江 杭州 311231
Author(s):
ZHU Xu-zhen*; YE Yong-ling; SONG Yuan-jiang; XIAO Zhi-gang; DAI Jian-ying.*
Traditional Chinese Medical Hospital of Hangzhou City,Hangzhou 310007,Zhejiang,China
關(guān)鍵詞:
干細(xì)胞 人脂肪干細(xì)胞 細(xì)胞外調(diào)節(jié)蛋白激酶 鎂黃長石
Keywords:
Stem cells;  Human adipose derived stem cell;  Extracellular regulated protein kinases;  Akermanite
摘要:
目的:探討鎂黃長石浸提液促進(jìn)人脂肪干細(xì)胞成骨分化的機(jī)制。方法:分離培養(yǎng)人脂肪干細(xì)胞,制備鎂黃長石浸提液母液及濃度為1%和10%的鎂黃長石浸提液。采用體外培養(yǎng)的第3代人脂肪干細(xì)胞分別進(jìn)行以下實(shí)驗(yàn):①將接種于鎂黃長石生物陶瓷材料上的第3代人脂肪干細(xì)胞分為2組,A組以生長培養(yǎng)液培養(yǎng)、B組以生長培養(yǎng)液+成骨誘導(dǎo)液培養(yǎng)。分別于培養(yǎng)開始前及培養(yǎng)開始后1、4、7 d和10 d提取培養(yǎng)液的上清液,采用電感耦合等離子體-原子發(fā)射光譜技術(shù)測定Ca、Mg、Si離子的濃度。②采用鎂黃長石浸提液母液+成骨誘導(dǎo)液培養(yǎng)第3代人脂肪干細(xì)胞,分別在培養(yǎng)開始后1、4、7、10、14、17、21 d以Western Blot法檢測細(xì)胞的細(xì)胞外調(diào)節(jié)蛋白激酶和磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶的表達(dá)情況,同時(shí)在培養(yǎng)開始后4、10、14、17 d測定堿性磷酸酶含量。③將培養(yǎng)的第3代人脂肪干細(xì)胞分為4組,Ⅰ組以生長培養(yǎng)液+成骨誘導(dǎo)液培養(yǎng)、Ⅱ組以1%鎂黃長石浸提液+成骨誘導(dǎo)液培養(yǎng)、Ⅲ組以10%鎂黃長石浸提液+成骨誘導(dǎo)液培養(yǎng)、Ⅳ組以鎂黃長石浸提液母液+成骨誘導(dǎo)液培養(yǎng)。培養(yǎng)至第10天時(shí),以Western Blot法檢測4組細(xì)胞的細(xì)胞外調(diào)節(jié)蛋白激酶和磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶的表達(dá)情況,并測定堿性磷酸酶定量含量。結(jié)果:①人脂肪干細(xì)胞體外培養(yǎng)過程中鎂黃長石析出離子濃度。培養(yǎng)過程中不同時(shí)間點(diǎn)的Ca離子濃度不同(P=0.001); 2組Ca離子濃度總體有差別(P=0.001),進(jìn)一步比較,A組各時(shí)間點(diǎn)Ca離子濃度均高于B組[0 d:(1.65±0.03)mmol·L-1,(1.51±0.01)mmol·L-1,P=0.001; 1 d:(4.78±0.18)mmol·L-1,(2.56±0.02)mmol·L-1,P=0.001; 4 d:(3.27±0.11)mmol·L-1,(1.90±0.01)mmol·L-1,P=0.001; 7 d:(3.24±0.03)mmol·L-1,(2.03±0.02)mmol·L-1,P=0.001; 10 d:(3.14±0.02)mmol·L-1,(2.13±0.03)mmol·L-1,P=0.000]; 時(shí)間因素與分組因素存在交互效應(yīng)(P=0.001)。培養(yǎng)過程中不同時(shí)間點(diǎn)的Mg離子濃度不同(P=0.001); 2組Mg離子濃度總體有差別(P=0.001),進(jìn)一步比較顯示,除0 d外,其余各時(shí)點(diǎn)A組Mg離子濃度均高于B組[0 d:(0.09±0.00)mmol·L-1,(0.09±0.01)mmol·L-1,P=0.407; 1 d:(0.46±0.02)mmol·L-1,(0.27±0.01)mmol·L-1,P=0.001; 4 d:(0.27±0.01)mmol·L-1,(0.13±0.01)mmol·L-1,P=0.001; 7 d:(0.26±0.01)mmol·L-1,(0.18±0.02)mmol·L-1,P=0.001; 10 d:(0.24±0.01)mmol·L-1,(0.17±0.01)mmol·L-1,P=0.001]; 時(shí)間因素與分組因素存在交互效應(yīng)(P=0.001)。培養(yǎng)過程中不同時(shí)間點(diǎn)的Si離子濃度不同(P=0.001); 2組Si離子濃度總體有差別(P=0.001),進(jìn)一步比較,除0 d外,其余各時(shí)點(diǎn)A組Si離子濃度均高于B組[0 d:(0.01±0.00)mmol·L-1,(0.01±0.00)mmol·L-1,P=1.000; 1 d:(2.52±0.02)mmol·L-1,(2.05±0.06)mmol·L-1,P=0.001; 4 d:(1.71±0.01)mmol·L-1,(0.53±0.01)mmol·L-1,P=0.001; 7 d:(1.91±0.01)mmol·L-1,(1.14±0.01)mmol·L-1,P=0.001; 10 d:(1.88±0.01)mmol·L-1,(1.24±0.01)mmol·L-1,P=0.001]; 時(shí)間因素與分組因素存在交互效應(yīng)(P=0.001)。②鎂黃長石浸提液作用時(shí)間對(duì)人脂肪干細(xì)胞成骨分化的影響。磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶在第7天開始表達(dá),第10天和第14天時(shí)表達(dá)量最高,第17天時(shí)表達(dá)量又降至基礎(chǔ)水平。第4天、第10天、第14天、第17天ALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(3.68±0.80)nmol PNP·min-1·mg-1,(7.90±0.50)nmol PNP·min-1·mg-1,(7.79±0.53)nmol PNP·min-1·mg-1,(6.93±0.86)nmol PNP·min-1·mg-1; P=0.000]。進(jìn)一步兩兩比較,第4天時(shí)ALP含量低于其余3個(gè)時(shí)間點(diǎn)(P=0.000),其余各時(shí)點(diǎn)兩兩比較,差異均無統(tǒng)計(jì)學(xué)意義。③鎂黃長石浸提液濃度對(duì)人脂肪干細(xì)胞成骨分化的影響。體外培養(yǎng)至第10天時(shí),各組細(xì)胞的細(xì)胞外調(diào)節(jié)蛋白激酶表達(dá)量基本相同,而磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶的表達(dá)量略有差異,其中Ⅱ組表達(dá)量最高。各組細(xì)胞ALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(5.26±0.25)nmol PNP·min-1·mg-1,(5.76±0.15)nmol PNP·min-1·mg-1,(5.32±0.20)nmol PNP·min-1·mg-1,(5.32±0.25)nmol PNP·min-1·mg-1; P=0.024]。進(jìn)一步兩兩比較,Ⅱ組含量高于其余3組(P=0.010,P=0.020,P=0.007),其余各組兩兩比較,差異均無統(tǒng)計(jì)學(xué)意義。結(jié)論:鎂黃長石浸提液可以促進(jìn)人脂肪干細(xì)胞成骨分化,且具有濃度依賴性,其機(jī)制可能與鎂黃長石析出的Ca、Mg、Si離子激活細(xì)胞外調(diào)節(jié)蛋白激酶轉(zhuǎn)導(dǎo)通路有關(guān)。
Abstract:
Objective:To explore the mechanism of extracts from akermanite promoting the human adipose derived stem cell(HADSC)osteogenetic differentiation in vitro.Methods:Separation culture was carried on HADSC,original extracts from akermanite and the extracts with concentrations of 1%and 10% were respectively prepared.The following experiments were respectively carried on the third generation of HADSC.①The cells inoculated on the akermanite bioceramics were divided into 2 groups,group A was cultured in growth media,group B was cultured in growth media combined with osteogenesis induced liquid.The supernatant of the culture solution was carried out before the culture and 1,4,7 and 10 days after the culture,then the concentrations of calcium ions,magnesium ions and silicium ions were detected through inductively coupled plasma atomic emission spectrometry(ICP-AES).②The cells were cultured in original extracts from akermanite combined with osteogenesis induced liquid.The contents of extracellular regulated protein kinases(ERK)and phosphorylation ERK were detected through Western Blot technique on 1,4,7,10,14,17 and 21 days after culture,and the contents of alkaline phosphatase(ALP)were detected on 4,10,14 and 17 days after culture.③The cells were divided into 4 groups,groupⅠwas cultured in growth media combined with osteogenesis induced liquid,groupⅡwas cultured in 1%extracts from akermanite combined with osteogenesis induced liquid,group Ⅲ was cultured in 10%extracts from akermanite combined with osteogenesis induced liquid,group Ⅳ was cultured in original extracts from akermanite combined with osteogenesis induced liquid.After culturing for 10 days,the contents of ERK and phosphorylation ERK were detected through Western Blot method,and the contents of ALP were detected.Results:①The concentrations of calcium ions were different at different time points in the process of culture(P=0.001).There was total difference in the concentrations of calcium ions between the 2 groups(P=0.001).Further multiple comparison indicated that the concentrations of calcium ions at different time points of group A were all higher than those of group B(0 d:1.65±0.03)mmol·L-1,(1.51±0.01)mmol·L-1,P=0.001; 1 d:(4.78±0.18)mmol·L-1,(2.56±0.02)mmol·L-1,P=0.001; 4 d:(3.27±0.11)mmol·L-1,(1.90±0.01)mmol·L-1,P=0.001; 7 d:(3.24±0.03)mmol·L-1,(2.03±0.02)mmol·L-1,P=0.001; 10 d:(3.14±0.02)mmol·L-1,(2.13±0.03)mmol·L-1,P=0.000).There was interaction effect between time factor and group factor(P=0.001).The concentrations of magnesium ions were different at different time points in the process of culture(P=0.001).There was total difference in the concentrations of magnesium ions between the 2 groups(P=0.001).Further multiple comparison indicated that the concentrations of magnesium ions at different time points of group A were all higher than those of group B except that on 0d[0 d:(0.09±0.00)mmol·L-1,(0.09±0.01)mmol·L-1,P=0.407; 1 d:(0.46±0.02)mmol·L-1,(0.27±0.01)mmol·L-1,P=0.001; 4 d:(0.27±0.01)mmol·L-1,(0.13±0.01)mmol·L-1,P=0.001; 7 d:(0.26±0.01)mmol·L-1,(0.18±0.02)mmol·L-1,P=0.001; 10 d:(0.24±0.01)mmol·L-1,(0.17±0.01)mmol·L-1,P=0.001].There was interaction effect between time factor and group factor(P=0.001).The concentrations of silicium ions were different at different time points in the process of culture(P=0.001).There was total difference in the concentrations of silicium ions between the 2 groups(P=0.001).Further multiple comparison indicated that the concentrations of silicium ions at different time points of group A were all higher than those of group B except that on 0 d[0 d:(0.01±0.00)mmol·L-1,(0.01±0.00)mmol·L-1,P=1.000; 1 d:(2.52±0.02)mmol·L-1,(2.05±0.06)mmol·L-1,P=0.001; 4 d:(1.71±0.01)mmol·L-1,(0.53±0.01)mmol·L-1,P=0.001; 7 d:(1.91±0.01)mmol·L-1,(1.14±0.01)mmol·L-1,P=0.001; 10 d:(1.88±0.01)mmol·L-1,(1.24±0.01)mmol·L-1,P=0.001].There was interaction effect between time factor and group factor(P=0.001).②Expression of phosphorylation ERK was started at the 7th day,and the contents of phosphorylation ERK reached the highest level at the 10th day and the 14th day,while the contents of phosphorylation ERK returned to the basic level at the 17th day.There was statistical difference in the contents of ALP among different time points(the 4th day:(3.68±0.80)nmol PNP·min-1·mg-1,the 10th day:(7.90±0.50)nmol PNP·min-1·mg-1,the 14th day:(7.79±0.53)nmol PNP·min-1·mg-1,the 17th day:(6.93±0.86)nmol PNP·min-1·mg-1; P=0.000).Further multiple comparison indicated that the contents of ALP at the 4th day was lower than that of the rest 3 time points(P=0.000),while there were no statistical differences between the other time points.③The contents of ERK for all the groups were the same basicly at the 10th day,while the contents of phosphorylation ERKof groupⅡwas the highest among the 4 groups.There was statistical difference in the contents of ALP among the 4 groups((5.26±0.25)nmol PNP·min-1·mg-1,(5.76±0.15)nmol PNP·min-1·mg-1,(5.32±0.20)nmol PNP·min-1·mg-1,(5.32±0.25)nmol PNP·min-1·mg-1; P=0.024).Further multiple comparison indicated that the contents of ALP of groupⅡwas higher than that of the rest 3 groups(P=0.010,P=0.020,P=0.007),while there were no statistical differences between the other groups.Conclusion:The extracts from akermanite can improve osteogenetic differentiation of HADSC with the feature of concentration dependency,and its mechanism may have relationships with ERK signal transduction pathway activated by calcium ions,magnesium ions and silicium ions from extracts from akermanite.

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更新日期/Last Update: 1900-01-01