长期低氧对骨肉瘤细胞增殖与分化的影响

Long-term hypoxia promotes proliferation and suppresses differentiation of osteosarcoma cells

目的观察长期低氧对骨肉瘤细胞增殖、分化及骨肉瘤干细胞自我更新能力的影响。方法采用噻吩蓝(MTT)比色法观察低氧对骨肉瘤细胞系MG63增殖的影响。后续试验分为两组:第1组为低氧处理组,将MG63细胞在低氧环境下培养1周后进行实验;第2组为常氧对照组。应用实时定量聚合酶链反应(real-timePCR)检测两组骨肉瘤细胞的成骨分化基因(Runx2和OC)、胚胎干细胞标志基因Oct3/4、原癌基因c-Myc及低氧诱导因子(HIF)-1α、2α的表达。采用无血清悬浮球培养法观察并比较两组细胞肉瘤球数目和体积的差异。采用免疫组织化学染色法检测两组细胞中HIF-1α和HIF-2α蛋白的表达。结果培养第6、9天,低氧处理组的光密度(D)值为0.128±0.020、0.700±0.060,分别显著高于常氧对照组的0.073±0.050、0.480±0.120(P值均<0.05)。低氧处理组MG63细胞骨肉瘤球的数目为(24±10.97)个,显著高于常氧对照组的(13±3.95)个(P<0.05);低氧处理组MG63骨肉瘤球的体积显著大于常氧对照组。与常氧对照组比较,低氧处理组细胞的成骨标志基因Runx2和OC均显著下调(P值均<0.05),胚胎干细胞标志基因Oct3/4和原癌基因c-Myc表达均显著上调(P值均<0.05),HIF-2α表达显著上调(P<0.05);免疫组织化学染色检测到MG63细胞中有HIF-1α和HIF-2α蛋白表达。结论长期低氧可促进骨肉瘤细胞MG63的增殖,抑制其成骨分化,并促进骨肉瘤球的自我更新能力,增强其干细胞特性。HIF-2α蛋白可能在这些过程中发挥关键作用。

Objective To determine the effect of long-term hypoxia on the proliferation,differentiation and self-renewal ability of osteosarcoma cells.Methods Osteosarcoma cell line MG63 was cultured under hypoxia（2%O2）for 7 d,or under normoxia for same period to serve as control.Cell proliferation was examined by 3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide（MTT）assay.The mRNA expressions of osteoblast differentiation genes Runx2 and OC,embryonic stem cells marker gene Oct3/4,proto oncogene c-Myc and hypoxia inducible factor（HIF）-1/2α was examined by real-time polymerase chain reaction（PCR）.Hypoxia-pretreated MG63 cells were cultured in serum-free medium to observe the number and size of sarcospheres.The protein expression of HIF-1/2α was detected by immunohistochemistry methods.Results After hypoxia culture for 6 and 9 d,the cell viability was higher in hypoxia group than in normoxia group（0.128±0.020 vs.0.073±0.050,and 0.700±0.060 vs.0.480±0.120,P0.05）,indicating that the proliferation of cells in hypoxia groups was significantly faster than normoxia groups.The number of sarcospheres formed in hypoxia groups was 24±10.97,significantly higher than in normoxia groups（13±3.95,P0.05）,and the size of sarcospheres in the former group were bigger than in the normoxia cells,suggesting hypoxia promoted self-renewal ability of osteosarcoma stem cells.The expressions of Runx2 and OC were suppressed（P0.05）,while those of Oct3/4,c-Myc and HIF-2α were up-regulated（P0.05）.The protein expressions of HIF-1α and HIF-2α were found by immunohistochemistry.Conclusion Long-term hypoxia promotes proliferation of osteosarcoma cells,suppresses osteogenic differentiation and improves stemness of osteosarcoma stem cells.HIF-2α may play an important role in these processes.