[1]朱旭贞,叶永玲,宋远江,等.镁黄长石浸提液促进人脂肪干细胞体外成骨分化的机制研究[J].中医正骨,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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镁黄长石浸提液促进人脂肪干细胞体外成骨分化的机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年05期
页码:
3-8
栏目:
基础研究
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:
朱旭贞1; 叶永玲1; 宋远江2; 肖治刚3; 戴建英4
1.浙江省杭州市中医院,浙江 杭州 310007; 2.浙江大学医学院附属第二医院, 浙江 杭州 310009; 3.上海交通大学医学院附属第九人民医院,上海 200011; 4.杭州市启迪生物有限公司,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
关键词:
干细胞 人脂肪干细胞 细胞外调节蛋白激酶 镁黄长石
Keywords:
Stem cells;  Human adipose derived stem cell;  Extracellular regulated protein kinases;  Akermanite
摘要:
目的:探讨镁黄长石浸提液促进人脂肪干细胞成骨分化的机制。方法:分离培养人脂肪干细胞,制备镁黄长石浸提液母液及浓度为1%和10%的镁黄长石浸提液。采用体外培养的第3代人脂肪干细胞分别进行以下实验:①将接种于镁黄长石生物陶瓷材料上的第3代人脂肪干细胞分为2组,A组以生长培养液培养、B组以生长培养液+成骨诱导液培养。分别于培养开始前及培养开始后1、4、7 d和10 d提取培养液的上清液,采用电感耦合等离子体-原子发射光谱技术测定Ca、Mg、Si离子的浓度。②采用镁黄长石浸提液母液+成骨诱导液培养第3代人脂肪干细胞,分别在培养开始后1、4、7、10、14、17、21 d以Western Blot法检测细胞的细胞外调节蛋白激酶和磷酸化细胞外调节蛋白激酶的表达情况,同时在培养开始后4、10、14、17 d测定碱性磷酸酶含量。③将培养的第3代人脂肪干细胞分为4组,Ⅰ组以生长培养液+成骨诱导液培养、Ⅱ组以1%镁黄长石浸提液+成骨诱导液培养、Ⅲ组以10%镁黄长石浸提液+成骨诱导液培养、Ⅳ组以镁黄长石浸提液母液+成骨诱导液培养。培养至第10天时,以Western Blot法检测4组细胞的细胞外调节蛋白激酶和磷酸化细胞外调节蛋白激酶的表达情况,并测定碱性磷酸酶定量含量。结果:①人脂肪干细胞体外培养过程中镁黄长石析出离子浓度。培养过程中不同时间点的Ca离子浓度不同(P=0.001); 2组Ca离子浓度总体有差别(P=0.001),进一步比较,A组各时间点Ca离子浓度均高于B组[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]; 时间因素与分组因素存在交互效应(P=0.001)。培养过程中不同时间点的Mg离子浓度不同(P=0.001); 2组Mg离子浓度总体有差别(P=0.001),进一步比较显示,除0 d外,其余各时点A组Mg离子浓度均高于B组[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]; 时间因素与分组因素存在交互效应(P=0.001)。培养过程中不同时间点的Si离子浓度不同(P=0.001); 2组Si离子浓度总体有差别(P=0.001),进一步比较,除0 d外,其余各时点A组Si离子浓度均高于B组[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]; 时间因素与分组因素存在交互效应(P=0.001)。②镁黄长石浸提液作用时间对人脂肪干细胞成骨分化的影响。磷酸化细胞外调节蛋白激酶在第7天开始表达,第10天和第14天时表达量最高,第17天时表达量又降至基础水平。第4天、第10天、第14天、第17天ALP含量比较,差异有统计学意义[(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]。进一步两两比较,第4天时ALP含量低于其余3个时间点(P=0.000),其余各时点两两比较,差异均无统计学意义。③镁黄长石浸提液浓度对人脂肪干细胞成骨分化的影响。体外培养至第10天时,各组细胞的细胞外调节蛋白激酶表达量基本相同,而磷酸化细胞外调节蛋白激酶的表达量略有差异,其中Ⅱ组表达量最高。各组细胞ALP含量比较,差异有统计学意义[(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]。进一步两两比较,Ⅱ组含量高于其余3组(P=0.010,P=0.020,P=0.007),其余各组两两比较,差异均无统计学意义。结论:镁黄长石浸提液可以促进人脂肪干细胞成骨分化,且具有浓度依赖性,其机制可能与镁黄长石析出的Ca、Mg、Si离子激活细胞外调节蛋白激酶转导通路有关。
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