摘要
探讨淫羊藿总黄酮(EF)促进人骨髓间充质干细胞(hBMSCs)成骨分化中的类雌激素作用。将第1代hBMSCs进行培养、传代、纯化,取第4代hBMSCs进行流式细胞仪检测细胞表面标志CD90和CD45;取第4~6代hBMSCs进行药物实验,CCK-8法检测不同浓度EF对hBMSCs增殖的影响;倒置相差显微镜下观察EF促进hBMSCs成骨分化的细胞形态;设置空白、EF、EF+ICI182780组分别作用hBMSCs,茜素红染色观察各组的钙结节形成情况,RT-PCR检测OPN、ALP基因的表达情况。结果提示,经传代、纯化的hBMSCs细胞表面CD90表达呈阳性,CD45表达呈阴性;低浓度的EF(2.5和5μg/mL)对hBMSCs增殖有促进作用,高浓度的EF(10、20、40μg/mL)对hBMSCs增殖有抑制作用;10μg/mL的EF作用于hBMSCs后,细胞在镜下呈现成骨细胞样形态;茜素红染色证明EF能促进钙结节的形成,而雌激素受体(ER)抑制剂ICI182780能拮抗EF的这种作用;EF能上调成骨相关基因OPN、ALP的表达(P<0.05),而ICI182780也能抑制EF的这种效用(P<0.05)。EF具有通过激活ER而促进hBMSC成骨分化的作用。
The purpose of the present study was to investigate the estrogic effect of total flavonoids of Epimedii Folium(EF)on the osteogenesis of human bone marrow mesenchymal stem cells(hBMSCs).The first generation of hBMSCs were cultured,subcultured and purified,and the fourth generation of hBMSCs were used to detect the cell surface markers CD90 and CD45 by flow cytometry.In the drug experiment,The effect of different concentrations of EF on the proliferation of hBMSCs was detected with the method of CCK-8.Three groups were set:Blank,EF and EF+ICI182780.Alizarin red staining was used to observe the formation of calcium nodules and RT-PCR was used to detect the expression of OPN and ALP gene.The results indicated that the CD90 expression on the surface purified hBMSCs cells was positive while the CD45 expression was negative.Low concentration of EF(2.5 and 5μg/mL)promoted the proliferation of hBMSCs,while high concentration of EF(10,20,40μg/mL)inhibited the proliferation of hBMSCs.The cells cultured with EF present an osteoblastic appearance under the microscope.Alizarin red staining showed that EF could promote the formation of calcium nodules,while estrogen receptor(ER)inhibitor ICI182780 could antagonize this effect of EF.EF could up-regulate the expression of osteoblast-associated gene OPN,ALP(P<0.05)and ICI182780 could inhibit the effect of EF(P<0.05).In conclusion,EF can promote the osteogenic differentiation of hBMSC by activating ER.
作者
林思文
谌少波
殷嫦嫦
邵银初
LIN Si-wen;CHEN Shao-bo;YIN Chang-chang;SHAO Yin-chu(1908 Hospital of the Joint Service of the Chinese people s Liberation Army,Nanchang 330001,China;Jiujiang University,Jiujiang 332000,China)
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2020年第3期373-378,共6页
Natural Product Research and Development
基金
江西省卫生计生委科技计划(20175572)。
关键词
淫羊藿总黄酮
HBMSCS
成骨分化
雌激素
total flavonoids of Epimedii Folium
hBMSCs
osteogenic differentiation
estrogen
