17β-雌二醇联合杜仲醇对SD大鼠骨髓间充质干细胞成骨分化的影响及机制分析  

Effect of 17β⁃estradiol combined with dulcitol on osteogenic differentiation of bone marrow mesenchymal stem cells in SD rats and analysis of the mechanism

作　　者：杨燕兵 苏红青 张玉宝[1] 庄忠宝 刘衡 YANG Yan-bing;SU Hong-qing;ZHANG Yu-bao;ZHUANG Zhong-bao;LIU Heng(Department of Orthopedics,Hengshui Second People's Hospital,Hengshui,Hebei Province 053000,China;College of Pharmacy,Hebei North University,Zhangjiakou,Hebei Province 075132,China)

机构地区：[1]衡水市第二人民医院骨二科,河北衡水053000 [2]河北北方学院药学院,河北张家口075132

出　　处：《解剖学研究》2025年第1期11-17,共7页Anatomy Research

基　　金：衡水市科技计划项目(2022014079Z)。

摘　　要：目的探讨17β-雌二醇(17β-E2)联合杜仲醇对大鼠骨髓间充质干细胞(BMSCs)成骨分化的影响,并阐明其成骨诱导的作用机制。方法通过全骨髓法分离提取SD大鼠BMSCs,连续传代3次,采用倒置光学显微镜观察传代培养BMSCs的细胞学形态。将第3代BMSCs SD大鼠细胞分成对照组、17β-E2组、杜仲醇组及联合诱导组,培养24 h后各组相应进行成骨诱导。采用CCK-8检测各组成骨诱导第1、2、3 d的BMSCs增殖活性;采用RT-PCR检测各组成骨诱导第7 d的碱性磷酸酶(ALP)、I型胶原蛋白(COL-1)、Runt相关转录因子2(Runx-2)、骨钙素(OCN)mRNA表达量;采用Western blot检测各组成骨诱导第7 d小窝蛋白(Cav1)/Hippo信号通路中Cav1、Hippo/转录共激活因子PDZ结合基序(TAZ)蛋白表达;于成骨诱导第7 d,采用二喹啉甲酸(BCA)检测各组ALP相对活性;采用茜素红染色检测各组成骨诱导第21 d的钙结节数量。结果对照组、17β-E2组、杜仲醇组和联合诱导组成骨诱导第1 d的细胞增殖活性分别为0.32±0.03、0.28±0.02、0.29±0.03和0.35±0.04,各组间差异无统计学意义(P>0.05);成骨诱导第2 d,17β-E2组、杜仲醇组及联合诱导组的细胞增殖活性(分别为0.57±0.03、0.55±0.04和0.58±0.05)均高于对照组(0.46±0.05)(P<0.05);成骨诱导第3 d,联合诱导组的细胞增殖活性(0.72±0.03)显著高于17β-E2组、杜仲醇组(分别为0.61±0.05、0.58±0.04)(P<0.05)。与对照组相比,17β-E2组、杜仲醇组及联合诱导组的ALP mRNA、COL-1mRNA、Runx-2 mRNA、OCN mRNA、ALP活性、钙结节数量、TAZ蛋白表达均更高,而Cav1蛋白表达均降低(P<0.05)。与联合诱导组相比,17β-E2组、杜仲醇组的ALP mRNA、COL-1 mRNA、Runx-2 mRNA、OCN mRNA、ALP活性、钙结节数量、TAZ蛋白表达均更低,而Cav1蛋白表达均升高(P<0.05)。结论17β-E2联合杜仲醇可显著提高SD大鼠BMSCs的ALP活性和钙盐沉积,上调ALP、COL-1、Runx-2、OCN等成骨分化相关指标的表达,从而更能�Objective To investigate the effects of 17β⁃estradiol(17β⁃E2)combined with dulcitol on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)and to elucidate the mechanism of ac⁃tion of osteogenic induction.Methods SD rat BMSCs were isolated and extracted by the whole bone marrow meth⁃od and passaged three times consecutively,and the cytological morphology of the passaged cultured BMSCs was ob⁃served using an inverted light microscope.The 3rd generation BMSCs SD rat cells were divided into control group,17β⁃E2 group,dulcitol group and coinduction group,and each group was cultured for 24 h for osteogenic induction accordingly.Proliferative activity of BMSCs in the 1st,2nd,and 3rd d of induction of each constituent bone was as⁃sayed using Cell Counting Kit⁃8(CCK⁃8).Alkaline phosphatase(ALP),collagen type I(COL⁃1),runt⁃related transcription factor 2(Runx⁃2),and osteocalcin(OCN)mRNA expression of each constituent on the 7th day of os⁃teoinduction were detected using real⁃time fluorescence quantitative polymerase chain reaction(RT⁃PCR).Protein blotting(Western blot)was used to detect Cav1,Hippo/transcriptional co⁃activator PDZ binding motif(TAZ)pro⁃tein expression in each component of the osteoinductive 7th d foveal protein(Cav1)/Hippo signaling pathway.On the 7th day of osteogenic induction,the relative activity of ALP in each group was detected by bicinchoninic acid(BCA);Alizarin red staining was used to detect the number of calcium nodules at 21 d after osteogenesis induction in each group.Results Comparison of the cell proliferation activity of each group(0.32±0.03,0.28±0.02,0.29±0.03 and 0.35±0.04 in control group,17β⁃E2 group,dulctitol group and coinduction group,respectively)on the 1st d of osteogenic induction,the difference was not statistically significant(P>0.05);on the 2nd d of osteogenic induc⁃tion,the cell proliferation activity of the 17β⁃E2 group,the dulcitol group,and the combined induction group(0.57±0.31,0.55±0.04 and 0.58±

关 键 词：17Β-雌二醇 杜仲醇 骨髓间充质干细胞 成骨分化 

分 类 号：R68[医药卫生—骨科学]