黄芪甲苷抑制COUP-TFII表达对成骨细胞分化的影响及相关分子机制研究  

Effect and Related Molecular Mechanism Study on Astragaloside Ⅳ Stimulating Osteoblast Differentiation by Inhibiting COUP-TFII Expression

作　　者：赵华桥[1] 朱红[1] 王家洪[1] 卿泉[1] 那江[1] 李斌[1] 杨东[1] ZHAO Huaqiao;ZHU Hong;WANG Jiahong(Department of Ankle, Mianyang Municipal Osteological Hospital （Mianyang Sichuan 621000, China)

机构地区：[1]绵阳市骨科医院足踝科,四川绵阳621000

出　　处：《四川中医》2018年第5期54-58,共5页Journal of Sichuan of Traditional Chinese Medicine

摘　　要：目的:探究黄芪甲苷对小鼠成骨前体细胞系MC3T3-E1细胞分化的影响及相关分子机制。方法:取小鼠胚胎成骨细胞MC3T3-E1经复苏后进行培养,再以成骨细胞分化培养基诱导分化后,应用倒置显微镜、茜素红染色等方法鉴定成骨细胞分化;取对数生长期的MC3T3-E1细胞,构建shRNA COUP-TFII的质粒载体,以慢病毒载体成功转染至MC3T3-E1的细胞,作为阳性对照组;取正常对数生长期细胞根据培养基中所加黄芪甲苷的浓度分为:空白对照组(0μg/ml)、黄芪甲苷低剂量组(25μg/ml)、中剂量组(50μg/ml)、高剂量组(100μg/ml);采用ALP活性检测和钙结节染色法评估黄芪甲苷对成骨细胞分化能力的影响;以荧光实时定量PCR(qRT-PCR)及蛋白免疫印迹(Western blot)法测定黄芪甲苷对MC3T3-E1细胞中COUP-TFII基因及蛋白表达的影响。结果:MC3T3-E1细胞培养18天后,细胞重叠生长,经茜红素染色可见大小形态不一的钙结节沉积;阳性对照组及黄芪甲苷处理组MC3T3-E1细胞中ALP活性及钙化结节程度均显著高于空白对照组,黄芪甲苷处理组间对比差异有统计学意义(P<0.05),阳性对照组与黄芪甲苷高剂量组无显著性差异(P>0.05);阳性对照组及黄芪甲苷处理组COUP-TFII基因及蛋白表达水平均显著低于空白对照组(P<0.05),黄芪甲苷处理组间对比差异有统计学意义(P<0.05),阳性对照组与黄芪甲苷高剂量组无显著性差异(P>0.05)。结论:黄芪甲苷能够通过抑制COUP-TFII表达,促进成骨细胞的分化。Objective： To explore the influence of astragaloside Ⅳ on differentiation of osteoblast precursor cell line MC3T3-E1 in mice and related molecular mechanisms. Methods： Mouse embryonic osteoblast cell MC3T3-E1 was cultured after recovery,and then induced and differentiated in osteoblast differentiation medium. Osteoblast differentiation was identified by using inverted microscope and alizarin red staining methods. MC3T3-E1 cells were selected at logarithmic growth stage,and plasmid vector of shRNA COUP-TFII was established. Lentiviral vectors were successfully transfected into MC3T3-E1 cells,as the positive controlled group. According to the concentration of astragaloside Ⅳ in culture medium,normal logarithmic growth phase cells were divided into the blank controlled group（ 0μg/ml）,astragaloside Ⅳ low-dose group（ 25μg/ml）,mid-dose group（ 50μg/ml）,high-dose group（ 100μg/ml）. The influence was evaluated of astragaloside Ⅳ on differentiation of osteoblasts by using ALP activity assay and calcium nodule staining method. The effect of astragaloside Ⅳ on COUP-TFII gene and protein expression in MC3T3-E1 cells was detected by using real-time fluorescence quantitative PCR（ qRT-PCR） and Western blot. Results： After MC3T3-E1 cell cultured 18 d,cells overlapped and grew with calcium nodules of different sizes and sizes after alizarin red staining. ALP activity and calcified nodules degree in MC3T3-E1 cells in positive controlled group and astragaloside treatment group were significantly higher than those in blank controlled group,with statistically significant difference in astragaloside treatment groups（ P〈0. 05）. There was no significant difference between the positive controlled group and high-dose astragaloside group（ P〉0. 05）. COUP-TFII gene and protein expression levels in positive controlled group and astragaloside treatment groups were significantly lower than those in the blank controlled group（ P〈 0. 05）. There was significant difference in astragaloside tre

关 键 词：黄芪甲苷 鸡卵清蛋白上游启动子转录因子Ⅱ 慢病毒载体 SHRNA 成骨细胞分化 

分 类 号：R285.5[医药卫生—中药学]