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  

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 

机构地区：[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

出　　处：《中医正骨》2013年第5期3-8,共6页The Journal of Traditional Chinese Orthopedics and Traumatology

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West-ern 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�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 bioceramies 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 cuhured 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, group Ⅰ was euhured in growth media combined with osteogenesis induced liquid, group Ⅱ was cultured in 1% extracts from akermanite combined with osteogenesis induced liquid, group m 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 calc

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 

分 类 号：R285.5[医药卫生—中药学]