摘要
目的观察不同氧浓度对小鼠骨髓细胞形成破骨样细胞的影响,探讨低氧对破骨细胞生成影响的分子机制。方法取6～9周龄KM小鼠骨髓细胞,用含1α,25(OH)_2D_3(10^(-8)mol/L)、地塞米松(10^(-8)mol/L)和10%胎牛血清的α-MEM培养基分别在氧浓度为20%(常氧组)、6%、3%和1%(均为低氧组)条件下培养。骨髓细胞纯化后加入第3～5代成骨细胞进行共培养,根据培养时间分组,每组样本数为4,在缺氧第5天进行实验,每项实验重复3次。用Trizol提取细胞总RNA;用半定量反转录PCR(RT-PCR)检测OPG、RANK及RANKL mRNA的表达;用Western blotting检测RANK蛋白的表达。结果与常氧组相比,3个低氧组OPG mRNA的表达均随氧浓度的降低而降低,RANK mRNA和RANK蛋白表达随氧浓度的降低而升高,RANKL mRNA的表达随氧浓度的降低而升高。结论低氧可促进破骨样细胞的生成,氧浓度为3%时破骨细胞生成达高峰。
Objective To investigate if the formation of mouse osteoclastic ceils is affected by hypoxia. Methods Mouse bone marrow cells were obtained from the tibiae of six-to-nine-week-old male mice, the cells were cultured in α- MEM culture medium containing 1α,25（OH）2D3 （10-Stool/L）, Dexamethasone （10-Smol/L） and 10% fetal bovine serum. After the bone marrow cells were purified, the 3 -5th generation osteoblast were added to. Co-incubation cells were divided into groups at random, each group had 4 samples, and then the cells were cultured for five days in normoxia （20% 02） and hypoxia （6% 02 , 3% 02 , 1% 02） respectively. The following experiments were done in fifth day and each experiment was repeated three times ： analysing the expression of OPG, RANK and RANKL mRNA by the method of reverse transcription polymerase chain reaction （RT-PCR）, detecting the expression of RANK protein by the method of western blotting. Results Oxygen concentration had significant effect on the formation of osteoelastic cells. With decrease of oxygen contents, compared with that of normoxia group, the expression of OPGmRNA in hypoxia groups decreased markedly; the expression of RANK mRNA and RANK protein increased; the expression of RANKL mRNA increased significantly. Conclusion Hypoxia increased the formation of mouse osteoclastic cells in vitro. With decrease of oxygen concentration, the number of the osteoclastic cells was gradually increased, especially in 3% 02.
出处
《中华骨质疏松和骨矿盐疾病杂志》
2010年第4期263-268,共6页
Chinese Journal Of Osteoporosis And Bone Mineral Research
