论文部分内容阅读
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。
OBJECTIVE: To investigate the effects of hypoxia on the expression of osteoprotegerin (OPG) and receptor activator of NF-κB in rat bone marrow derived mesenchymal stem cells (rBMSCs) ligand, RANKL) mRNA expression. Methods: The rBMSCs were isolated and cultured by whole bone marrow adherent method. Hypoxic model was established by using cobalt chloride (CoCl_2) as chemical hypoxia, and the cells were incubated with CoCl 2 at concentrations of 0, 50, 100, 200 and 400μmol / L, The effects of CoCl_2 on cell proliferation were detected by MTT assay. The expression of hypoxia inducible factor-1α (HIF-1α) was detected by real-time fluorescence quantitative PCR and Western blot. rBMSCs were treated with hypoxia for 0, 12, 24, 48, 72, 96 hours. The expression of OPG and RANKL mRNA was detected by real-time fluorescence quantitative PCR. SPSS18.0 software package for statistical analysis of the data. Results: Compared with the control group, CoCl 2 at 200 and 400 μmol / L inhibited the proliferation of rBMSCs (P <0.05), while the proliferation at 50 and 100 μmol / L of CoCl 2 did not affect significantly (P> 0.05). The mRNA and protein levels of HIF-1α in rBMSCs were higher in 50,100μmol / L CoCl_2 group than those in 100μmol / L CoCl_2 group compared with 50μmol / L CoCl_2 group. Compared with normoxia group, the expression of OPG and RANKL mRNA in rBMSCs of hypoxia group and control group did not change after incubation with 100 μmol / L CoCl 2 for 12 h (P> 0.05). At 24, 48, 72, 96 h, Group OPG mRNA expression increased, RANKL mRNA expression decreased, OPG / RANKL ratio was significantly increased (P <0.05). Conclusion: Hypoxia treatment with 100 μmol / L CoCl 2 can promote osteogenic differentiation by regulating the expression of OPG and RANKL mRNA in rBMSCs.