摘要
目的:探讨模拟低氧环境对人乳腺癌MCF-7细胞增殖及低氧诱导因子(HIF-1α)、基质衍生因子-1(SDF-1)和趋化因子受体4(CXCR4)表达的影响。方法:常规复苏、传代培养MCF-7细胞,待细胞对数期生长后分别用50、100、150和200μmol/L CoCl2处理细胞,并于12、24和48h收集细胞进行以下指标检测。MTT比色法检测细胞增殖抑制率;RT-PCR检测HIF-1α、SDF-1和CXCR4的mRNA表达;蛋白质印迹法检测HIF-1α和CXCR4在蛋白水平的表达;免疫荧光法测定150μmol/L CoCl2经不同时间处理后MCF-7细胞中SDF-1的表达。结果:低氧模拟微环境中,应用CoCl2处理MCF-7细胞后有较明显抑制作用,实验中以CoCl2(50、100、150和200μmol/L)处理MCF-7细胞24h后抑制率分别为(54.62±0.07)%、(65.21±0.03)%、(80.15±0.01)%和(94.51±0.01)%;以CoCl2150μmol/L浓度处理细胞12、24和48h后,抑制率分别为(70.83±0.03)%、(80.15±0.01)%和(89.27±0.03)%;RT-PCR和蛋白质印迹法检测结果表明,HIF-1α、SDF-1和CXCR4的mRNA的表达及HIF-1α和CXCR4蛋白的表达与低氧时间和CoCl2浓度有关,以低氧处理细胞24h及CoCl2浓度150μmol/L时最明显;免疫荧光结果表明CoCl2150μmol/L时MCF-7细胞SDF-1蛋白的表达随时间延长有增加。结论:CoCl2模拟低氧后,细胞生长受抑制呈时间和浓度依赖性;CoCl2浓度在50～150μmol/L时,HIF-1α、SDF-1和CXCR4在mRNA和蛋白水平表达上调并呈现时间和浓度依赖性。
OBJECTIVE： To investigate the effects of hypoxia on the proliferation of human breast cancer MCF-7 cells and the cellular expression of hypoxia inducible factor-la, stromal cell derived factor-1 and CXC chemokine receptor 4. METHODS： Human breast cancer MCF-7 cells with exponential growth in routine culture were exposed to 50,100,150 and 200 μmol/L CoCl2 to mimic hypoxic conditions,At 12,24 and 48 h,the cells were collected for MTT assay,RT-PCR for HIF-1a, SDF-1 and CXCR4 mRNAs expression,Western bolt for HIF-1a and CXCR4 proteins expression,and the ex- pression of SDF-1 in MCF-7 cells exposed to hypoxia （by COC12 150 μmol/L） at different time points were detected by im- munofluorescence. RESULTS： Compared with the cells without COC12 treatment,a significant growth inhibition which in- creased with CoCl2 concentration and exposure time was observed, the inhibition rate of 50,100 and 150 μmol/L with COC12 for 24 hwas （54. 62±0.07）%,（65.21±0.03）%,（80. 15±0.01）% and （94. 51±0.01）%,respectively. With 150 μmol/L COC12 for 12,24 and 48 h, the inhibition rate were （70. 83±0.03）%, （80. 15±0.01）% and （89.27±0.03） % ,respectively. RT-PCR showed that over-expression of HIF-1a, SDF-1 and CXCR4 mRNAs and western bolt- showed that over-expression of HIF-la and CXCR4 proteins,which could be measured under hypoxia induced by CoCl2.The immunofluorescence showed that the expression of SDF-1 protein increased with time extension after the cells stimu- lated by 150 μmol/L CoCl2. CONCLUSION： Hypoxia mimiced by CoClz exposure significantly inhibits the proliferation of MCF-7 cells, and at the non-toxic doses, CoCl2 dose-and time-dependently increases the expression of HIF-1 a, SDF-1 and CXCR4.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2013年第12期904-908,共5页
Chinese Journal of Cancer Prevention and Treatment
基金
湖南省卫生厅一般项目(B2010-039)
关键词
乳腺肿瘤
低氧诱导因子
基质衍生因子-1
趋化因子受体4
二氯化钴
breast neoplasms
hypoxia inducible factor
stromal cell derived factor 1
cxc chemokine receptor 4
co- baltous chlorid
