氯化钴诱导建立口腔鳞状细胞癌低氧模型及其生物学行为初探

Establishment of CoCl2 induced hypoxic cell model of oral squamous cell carcinoma and a preliminary study on the biological behaviour change

目的 利用氯化钴（CoCl2）化学试剂模拟口腔鳞状细胞癌细胞低氧模型,探讨低氧环境对癌细胞生物学行为的影响.方法 用50、100、150、200 μmol/L的CoCl2化学试剂处理口腔鳞状细胞癌细胞株HSC-3及SCC-4,同时分为对照组（不含CoCl2的DMEM培养液培养细胞）和实验组（SCC-4、HSC-3细胞经不同浓度CoCl2处理）.通过荧光定量PCR方法,首先在mRNA水平检测乏氧诱导因子1 α（hypoxia inducible factor-1α,HIF-1α）及下游靶基因血管内皮生长因子（vascular endothelial growth factor,VEGF）、B淋巴细胞瘤2（B-cell lymphoma-2,BCL-2）的表达,并用蛋白质印迹法进一步验证;通过细胞增殖及流式细胞术检测低氧对口腔鳞状细胞癌生长、凋亡及细胞周期的影响.采用细胞划痕及Transwell等检测细胞迁移能力的改变.结果 与对照组相比,150 μmol/L CoCl2培养24 h后,实验组SCC-4细胞内HIF-1α、VEGF及BCL-2的mRNA表达水平分别增加6.00±0.20、5.40±0.40和5.40±0.30;HSC-3细胞分别增加5.60±0.30、5.20±0.60、5.80±0.40.150 μmol/L的CoCl2培养2～3d后,HSC-3、SCC-4实验组与对照组相比,细胞生长速度降低,结果经t检验,P值均小于0.05;流式细胞术检测发现,150 μ.mol/L CoCl2培养后的细胞凋亡增加,对照组HSC-3细胞早期凋亡＋晚期凋亡的比例为2.25％,实验组为5.82％;对照组SCC-4细胞早期凋亡＋晚期凋亡的比例是2.58％,而实验组为10.27％.Transwell实验中对照组HSC-3和SCC-4细胞24 h迁移的面积占比分别为（31.5±2.3）％、（29.1±1.5）％,实验组该比率分别为（18.3±1.9）％、（13.2±0.8）％,两组比较差异有统计学意义（P＜0.05）.结论 利用化学诱导剂CoCl2可在常氧条件下诱导出口腔鳞状细胞癌的低氧模型,肿瘤细胞内低氧标志物表达均明显提高,同时细胞生物学行为也发生改变,细胞增殖速度降低,凋亡增加,迁移速度降低.

Objective To mimic oral squamous cell carcinoma（OSCC） cell hypoxia by using chemical agent CoCl2 and to investigate its biological behaviour.Methods Oral squamous cell carcinoma cell lines HSC-3 and SCC-4 were exposed to different concentration of CoCl2.HSC-3 and SCC-4 cells were treated with 50,100,150,200 μ mol/L CoCl2.Expression of hypoxia inducible factor-1α（HIF-1α）,vascular endothelial growth factor（VEGF） and B-cell lymphoma-2（BCL-2） were measured by real time polymerase chain reaction（PCR） and Western blotting in both mRNA and protein level.Cell proliferation,cell apoptosis and cell cycle were detected to analyze its biological behaviour.Both wound healing and Transwell assay were applied to test the ability of cell igration.Results The result showed that after treatment of 150 μ mol/L CoCl22 for 24 h,mRNA level of HIF-1α,VEGF and Bcl-2 was increased by 6.00±0.20,5.40±0.40,5.40±0.30 （SCC-4）; 5.60±0.30,5.20±0.60,5.80±0.40（HSC-3）.OSCC cells treated with 150 μmol/L CoCl2 for 24 h were collected.Compared with control group,the growth rate of cells was significantly decreased,P value was less than 0.05 （when HSC-3,SCC-4 cultured for 2 and 3 days）.The apoptosis of OSCC cells was increased when treated with 150 μmol/L CoCl2 for 24 h：HSC-3 2.25%（control group） and 5.82%（treatment group）; SCC-4 2.58%（control group） and 10.27% （treatment group）.The migration ablility of OSCC cells was decreased when using 150 μmol/L CoCl2 for 24 h.The migration area ratio was（31.5±2.3） %（HSC-3）,（29.1± 1.5） % （SCC-4） in control group and（18.3± 1.9） % （HSC-3）,（13.2±0.8）% （SCC-4） in treatment group（P〈0.05）.Conclusions The hypoxic cell model of OSCC could be induced by CoCl2.The expression level of hypoxic markers was up regulated significantly and the cells biological behaviour changed including decreased cell proliferation,increased apoptosis and decreased migration.