不同氧环境中破骨细胞特异性基因的表达

Specific gene expression of osteoclasts under different oxygen tension

背景:课题组以往研究证实,氧浓度过低乃至处于低氧时(体积分数2%O2),破骨前体细胞的增殖及破骨细胞的分化和功能都受到抑制,但体外培养的破骨细胞在不同氧环境下的基因表达未见有相关报道。目的:实验拟观察不同氧环境下破骨细胞各特异性基因的表达规律,探寻氧环境改变影响破骨细胞分化的表达机制。方法:将破骨前体细胞株经质量浓度均为10μg/L的核因子κB受体活化因子配体和巨噬细胞集落刺激因子联合诱导成为成熟的破骨细胞,然后分别置于常氧、组织氧、低氧(体积分数20%,7%,2%)条件下培养,用抗酒石酸酸性磷酸酶染色检测破骨细胞形成的变化,并分别在培养第1-7天收集细胞,通过定量PCR方法检测核因子κB受体活化因子,肿瘤坏死因子受体相关因子6,抗酒石酸酸性磷酸酶,组织蛋白酶K基因mRNA的表达。结果与结论:低氧条件下抗酒石酸酸性磷酸酶阳性的破骨细胞数显著低于组织氧和常氧培养时形成的抗酒石酸酸性磷酸酶阳性细胞数(P<0.05)。不同氧环境下,破骨细胞中核因子κB受体活化因子mRNA的表达无明显改变,组织氧和常氧培养下肿瘤坏死因子受体相关因子6 mRNA的表达在培养第5天最高(P<0.05)。随着氧浓度的降低,抗酒石酸酸性磷酸酶和组织蛋白酶K mRNA表达时间延后,组织氧培养条件下此2者的表达可以保持在最高水平。结果证实,与常氧和低氧条件相比,破骨细胞在组织氧培养条件下培养时更易促进其分化,从而维持其活性和功能。

BACKGROUND： Preliminary studies of our research group have confirmed that the proliferation of preosteoclasts and the differentiation and function of osteoclasts could be inhibited when they were cultured in lower oxygen tension even hypoxia （2% 02）, but the gene expression of osteoclasts cultured in vitro have not been reported. OBJECTIVE： To examine the effect of oxygen tension on specific gene expression of osteoclasts in vitro and explore the mechanism of osteoclast differentiation influenced by oxygen tension. METHODS： The preosteoclasts were induced with 10 μg/L macrophage colony stimulating facto and 10 μg/L soluble receptor activator of nuclear factor-KB ligand into mature osteoclasts. Then the osteoclasts were cultured in normoxia, tissue oxygen and hypoxia （20%, 7%, 2% 02） respectively. Cells were then stained for tartarate-resistant acid phosphatase to assess osteoclastic formation. Cells were collected at 1, 2, 3, 4, 5, 6, 7 days after culture respectively. The soluble receptor activator of nuclear factor-KB ligand, tumor necrosis factor receptor-associated factor 6, tartarate-resistant acid phosphatase, and cathepsin K mRNA expression levels were determined using real-time quantitative PCR. RESULTS AND CONCLUSION： The number of osteoclasts positive for tartarate-resistant acid phosphatase in the hypoxia was significantly lower than that in the tissue oxygen and normoxia （P 〈 0.05）. Under different oxygen tension, the mRNA expression levels of soluble receptor activator of nuclear factor-KB ligand in osteoclasts maintained unchanged. The mRNA expression levels of tumor necrosis factor receptor-associated factor 6 reached the peak at 5 days after culture in tissue oxygen and normoxia （P 〈 0.05）. The mRNA expression time of tartarate-resistant acid phosphatase and Cathepsin K were delayed accompanied by decreased oxygen tension, but the maximum were maintained in tissue oxygen. Compared with normoxia and hypoxia, osteoclasts cultured in tissue oxygen are more prone to differentiate and maintain the activity and functions.