中文摘要：

目的：通过低氧刺激乳腺癌MCF-7细胞,比较常氧与低氧条件下获取的MCF-7细胞条件培养液对成骨前体细胞MC3T3-E1分化能力的影响,并初步探讨信号素3A（semaphorin 3A,Sema3A）表达对低氧调控乳腺癌骨转移中成骨细胞分化的作用。方法：收集乳腺癌MCF-7细胞在常氧和低氧条件下培养48 h后的条件培养液,用其培养成骨前体细胞MC3T3-E1。采用BCIP/NBT碱性磷酸酶（alkaline phosphatase,ALP）显色试剂盒和ALP活性检测试剂盒检测MC3T3-E1细胞中ALP的含量及活性,茜素红S（alizarin red S,ARS）染色法检测MC3T3-E1细胞的矿化情况。同时,采用实时荧光定量PCR法和蛋白质印迹法检测常氧和低氧条件培养0、12、24和48 h后MCF-7细胞中Sema3A的表达变化。将重组人Sema3A蛋白加入到MCF-7低氧条件培养液处理的MC3T3-E1细胞中,实时荧光定量PCR法检测各组细胞中成骨分化相关蛋白ALP、Runt相关转录因子2（Runt-related transcription factor 2,RUNX2）、骨钙蛋白（osteocalcin,OCN）和转录因子Osterix（Osx）的m RNA表达水平。结果：相比于常氧条件培养获得的MCF-7条件培养液组,低氧条件培养获得的MCF-7条件培养液可以使MC3T3-E1细胞中ALP的活性明显减弱（P〈0.05）,且ARS染色钙化面积明显减小（P〈0.05）。低氧处理24和48 h后,MCF-7细胞中Sema3A的表达水平明显低于常氧条件下相同时间点的表达水平（P值均〈0.05）。向低氧条件培养液培养的MC3T3-E1细胞中加入重组人Sema3A蛋白处理后,MC3T3-E1细胞中ALP、RUNX2、OCN和Osx m RNA的表达水平均较未加重组人Sema3A处理组明显升高（P值均〈0.05）。结论：体外低氧环境可下调乳腺癌MCF-7细胞中Sema3A的表达,进而抑制成骨细胞的分化。提示Sema3A可能是低氧环境下乳腺癌条件培养液抑制成骨细胞分化的重要因素之一。

英文摘要：

Objective： To compare the effect of MCF-7 conditioned medium under normoxia or hypoxia condition on the differentiation of osteoblastic precursor MC3T3-E1 cells after exposing breast cancer MCF-7 cells to hypoxia, and to investigate the role of semaphorin 3A （Sema3A） on osteogenic differentiation regulated by hypoxia in breast cancer with bone metastasis. Methods： The conditioned medium was collected from breast cancer MCF-7 cells cultured under the normoxia or hypoxia condition for 48 h, and used to incubate osteoblastic precursor MC3T3-E1 cells. The content and activity of alkaline phosphatase （ALP） in MC3T3-E1 cells were evaluated by BClP/NBT ALP color development kit and ALP activity assay kit, respectively. The mineralization of MC3T3-E1 cells was detected by alizarin red S （ARS） staining. The expressions of Sema3A mRNA and protein in MCF-7 cells under normoxia or hypoxia condition for 0, 12, 24 and 48 h were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The recombinant human Sema3A （rhSema3A） was added into MC3T3-E1 cells incubated with MCF-7 hypoxia conditioned medium, then the mRNA expression levels of osteogenic differentiation-related proteins ALP, Runt-related transcription factor 2 （RUNX2）, osteocalcin （OCN） and Osterix （Osx） in MC3T3-E1 cells were measured by real-time fluorescent quantitative PCR. Results： Compared with normoxia cultured MCF-7 conditioned medium （NorCM） group, the activity of ALP in MC3T3-E1 cells was significantly decreased in hypoxia cultured MCF-7 conditioned medium （HyCM） group （P 〈 0.05）, and the area of mineralization nodes stained with ARS was also markedly reduced in HyCM group （P 〈 0.05）. The mRNA and protein expressions of Sema3A in MCF-7 cells after hypoxia culture for 24 and 48 h were significantly lower than those after normoxia culture for the same time （all P 〈 0.05）. After rhSema3A was used to treat MC3T3-E1 cells which were incubated with HyCM, the mRNA expression le