氯化钴低氧诱导食管癌细胞VEGF与per2的节律性研究

Relationship between circadian expression of per2 and VEGF induced by CoCl_2 in esophageal cancer cells

目的鉴定氯化钴(CoCl2)诱导离体人食管癌细胞株Eca-109化学性低氧的最佳浓度。检测离体人食管癌细胞株Eca-109在持续黑暗与正常光暗循环条件下per2与血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的节律相位。方法 cck-8检测0、50、100、150、200、250、300和400μmol/L CoCl2诱导人食管癌细胞化学性低氧的情况。定量PCR检测per2与VEGF基因的节律相位。结果不同浓度CoCl2对人食管癌细胞增殖性影响不同。人食管癌细胞株Eca-109无论在持续黑暗还是正常光暗循环条件下,per2与VEGF的表达均成自发节律性变化,per2的表达高低峰分别位于19:00和7:00,t19:00=60.62,t7:00=-20.27,P值均<0.05;VEGF高低峰位于3:00和19:00,t3:00=-13.86,t19:00=-30.00,P值均<0.05。结论 CoCl2在150μmol/L浓度下诱导人食管癌细胞化学性低氧效果最佳,CoCl2在高浓度250、300和400μmol/L对人食管癌细胞的增殖有明显的抑制作用。无论持续黑暗还是正常光暗循环,并不会影响人食管癌细胞节律基因per2与VEGF的表达,per2对VEGF的表达呈负性调节作用。

OBJECTIVE To identify the best concentration of COC12 for inducing hypoxia on in vitro human esophageal cancer cells Eca-109 ,test the circadian phase of per2 and VEGF^s expression when Eca-109 ceils in vitro under dark condition and cycled light-dark condition. METHODS Using cck-8 to test the Eca-109 celPs chemical hypoxia in different concentration of COC12. Using dark low-oxygen condition and low-oxygen condition as experimental group and control group in the culture of Eca-109 cells in vitro to test the circadian phase of per2 and VEGF by real-time quantitative PCR. RESULTS Different concentration of CoClz （0,50,100,150,200,250,300 and 400μmol/L） had different influence on the hyperplastic toxicity of the Eca-109 cells. The expression of per2 and VEGF was spontaneously rhythmic whether under continuous dark or cycled light-dark condition. The time for per2 reaching the maximum and minimum expression were 19100, 7100 （t19,00 =60. 62,t7,00 =-20. 27,P〈20. 05）. The time /or VEGF reaching the maximum and minimum expres- sion were 3100,19100 （t3,00 = -13.86,t19,00 = --30.00,P〈0.05）. CONCLUSIONS The hypoxic effect induced by COC12 is the best in 150μmol/L concentrations, there is obvious inhibition for Eca-109 cell＇s hyperplasia in high concentration of CoClz （250,300,400 μmol/L）. The dark or cycled light-dark condition has no impact on the expression of per2 and VEGF. Per2 has a down-regulation effect on VEGF＇s expression.