低氧调控大鼠骨髓间充质干细胞OPG/RANKL mRNA的表达

doi:10.19439/j.sjos.2017.03.005

上海口腔医学 ›› 2017, Vol. 26 ›› Issue (3): 258-262.doi: 10.19439/j.sjos.2017.03.005

低氧调控大鼠骨髓间充质干细胞OPG/RANKL mRNA的表达

史新连¹, 胡碧波², 任曼曼¹, 喻文彬², 邓辉¹

1.温州医科大学附属口腔医院牙周病科,2.正畸科,浙江温州 325027

收稿日期:2016-12-02 修回日期:2017-01-24 出版日期:2017-06-25 发布日期:2017-07-05
通讯作者: 邓辉,E-mail：dh0726@gmail.com
作者简介:史新连（1992-）,女,硕士研究生,E-mail：472277916@qq.com
基金资助:
浙江省自然科学基金（LY17H140009）; 浙江省大学生科技创新活动计划暨新苗人才计划（2016R413074）; 浙江省医药卫生科技计划（2015KYA149）; 温州市公益性科技计划（Y20160140）

Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells

SHI Xin-lian¹, HU Bi-bo², REN Man-man¹, YU Wen-bin², DENG Hui¹

1.Department of Periodontology,
2.Department of Orthodontics, School of Stomatology, Wenzhou Medical University. Wenzhou 325027, Zhejiang Province, China

Received:2016-12-02 Revised:2017-01-24 Online:2017-06-25 Published:2017-07-05

摘要/Abstract

摘要： 目的探讨低氧处理对大鼠骨髓间充质干细胞（rat bone marrow derived mesenchymal stem cells, rBMSCs）骨保护素（osteoprotegerin,OPG）和核因子κb受体活化因子配体（receptor activator of NF-κb ligand,RANKL）mRNA表达的影响。方法采用全骨髓细胞贴壁法分离、培养rBMSCs,应用化学低氧剂氯化钴（CoCl₂）建立低氧模型,分别以0、50、100、200、400 μmol/L浓度的CoCl₂孵育细胞,首先采用MTT法检测CoCl₂对细胞增殖的影响;利用实时荧光定量PCR及Western免疫印迹检测低氧诱导因子1α（hypoxia inducible factor -1α, HIF-1α）的表达情况。rBMSCs经低氧处理0、12、24、48、72、96 h,实时荧光定量PCR检测OPG、RANKL mRNA的表达。采用SPSS18.0软件包对数据进行统计学分析。结果与对照组相比,200、400 μmol/L的CoCl₂抑制rBMSCs增殖（P<0.05）,而50、100 μmol/L CoCl₂实验组的增殖并未产生显著影响（P>0.05）。在50、100 μmol/L CoCl₂实验组,rBMSCs表达HIF-1α 的mRNA和蛋白水平均高于对照组,100 μmol/L CoCl₂组较50 μmol/L CoCl₂组高。100 μmol/L CoCl₂孵育12 h时,低氧组和对照组rBMSCs的OPG、RANKL mRNA的表达无变化（P>0.05）; 24、48、72、96 h时,与常氧组相比,低氧组OPG mRNA表达水平升高, RANKL mRNA表达水平下降,OPG/RANKL的比值显著升高（P<0.05）。结论100 μmol/L CoCl₂低氧处理可通过调控rBMSCs OPG、RANKL mRNA的表达,从而促进成骨分化。

关键词: 大鼠骨髓间充质干细胞, 低氧, 骨保护素, 核因子κ, b受体活化因子配体

Abstract: PURPOSE: To investigate the effects of hypoxia on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNA in rat bone marrow mesenchymal stem cells (rBMSCs). METHODS: rBMSCs were isolated and cultured by whole bone marrow cell adherent method, and an optimal hypoxic preconditioning model was established with CoCl₂ (cobalt chloride). rBMSCs were incubated in cell culture mediums with different concentrations of CoCl₂(final concentrations of CoCl₂ were 0, 50, 100, 200, 400 μmol/L) and incubated for different times. MTT assay was applied to detect the effect of CoCl₂ on cell proliferation. mRNA and protein expression of HIF-1α of rBMSCs was detected by real-time PCR and Western blot. After treated with 100 μmol/L CoCl₂ for 0, 12, 24, 48, 72, 96 h, the expression of rBMSCs OPG/RANKL mRNA were detected by real-time PCR. The differences in distribution of each genotype were analyzed with SPSS 18.0 software package. RESULTS: Compared with the control group, 200, 400 μmol/L CoCl₂ inhibited the proliferation of rBMSCs (P<0.05). However, 50, 100 μmol/L CoCl₂ had no significant impact on the proliferation of rBMSCs (P>0.05). Real-time PCR and Western blot showed that HIF-1α expression in 50 μmol/L and 100 μmol/L CoCl₂ groups was significantly higher than the control group; the effect of 100 μmol/L CoCl₂ was significantly greater than 50 μmol/L CoCl₂. After cultivated in hypoxia condition for 12 h, the expression of OPG and RANKL mRNA in rBMSCs didn't change significantly (P>0.05). After cultured hypoxia condition for 24, 48, 72, 96 h, the expression of OPG mRNA in rBMSCs increased while the RANKL decreased, thus the ratio of OPG/RANKL increased and the difference was significant

Key words: Rat bone marrow derived mesenchymal stem cells, Hypoxia, Osteoprotegerin, Receptor activator of NF-κb ligand

中图分类号:

R781.4

史新连, 胡碧波, 任曼曼, 喻文彬, 邓辉. 低氧调控大鼠骨髓间充质干细胞OPG/RANKL mRNA的表达[J]. 上海口腔医学, 2017, 26(3): 258-262.

SHI Xin-lian, HU Bi-bo, REN Man-man, YU Wen-bin, DENG Hui. Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells[J]. Shanghai Journal of Stomatology, 2017, 26(3): 258-262.

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低氧调控大鼠骨髓间充质干细胞OPG/RANKL mRNA的表达

Hypoxia regulates the expression of OPG/RANKL mRNA in rat bone marrow mesenchymal stem cells

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