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

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(CoCl_2)建立低氧模型,分别以0、50、100、200、400μmol/L浓度的CoCl_2孵育细胞,首先采用MTT法检测CoCl_2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、400μmol/L的CoCl_2抑制rBMSCs增殖(P<0.05),而50、100μmol/L CoCl_2实验组的增殖并未产生显著影响(P>0.05)。在50、100μmol/L CoCl_2实验组,rBMSCs表达HIF-1α的mRNA和蛋白水平均高于对照组,100μmol/L CoCl_2组较50μmol/L CoCl_2组高。100μmol/L CoCl_2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:100μmol/L CoCl_2低氧处理可通过调控rBMSCs OPG、RANKL mRNA的表达,从而促进成骨分化。

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 stern 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 μmo/L CoC12 groups was significantly higher than the control group; the effect of 100 μmol/L CoC12 was significantly greater than 50 μmol/L CoC12 . 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 （P〈0.05）. CONCLUSIONS： Hypoxia can regulate the mRNA expression of OPG and RANKL mRNA in rBMSCs and significantly promote osteogenic differentiation.