喜树碱对低氧诱导HaCaT细胞趋化因子配体20表达的影响

Effects of camptothecin on the expression of CCL20 in HaCaT cells under hypoxia

目的观察喜树碱(CPT)对低氧(2%O2)培养下人永生化角质形成细胞(HaCaT细胞)趋化因子配体(CCL20)表达的影响,探讨CPT治疗斑块状银屑病可能的作用机制。方法将HaCaT细胞分为常氧组(21%O2点)和低氧(2%O2点)组,培养12 h,实时荧光定量聚合酶链反应(RT-PCR)检测CCL20 mRNA 的相对表达,酶联免疫吸附试验(ELISA)Kit测定细胞培养上清中CCL20表达;将12.5、25.0、50.0、100.0、200.0 nmol/L的CPT作用低氧诱导12 h的HaCaT细胞,ELISA Kit测定细胞培养上清中CCL20表达。结果常氧组和低氧组培养12 h,HaCaT细胞CCL20 mRNA 表达(ΔCT值)分别为-15.19±0.13和-13.70±0.10,两组间表达差异有统计学意义(t=-14.430,P=0.001);低氧培养12 h后HaCaT细胞(1×105个细胞)较常氧组CCL20蛋白表达增多,分别为(112.18±28.66)pg/ml、(64.36±47.85)pg/ml,但两组间表达差异无统计学意义(t=-1.485,P=0.212);100.0、200.0 nmol/L CPT处理低氧诱导12 h的HaCaT细胞(1×105个细胞)CCL20的表达分别为(64.35±19.70)pg/ml、(74.35±23.85)pg/ml,溶媒对照组为(112.18±28.66)pg/ml,组间多重比较差异有统计学意义(P<0.05)。结论低氧可诱导HaCaT细胞CCL20 mRNA 表达增加;100.0、200.0 nmol/LCPT可抑制HaCaT细胞CCL20蛋白的表达。

Objective To investigate the infl uence of camptothecin（CPT） on the expressions of CCL20 in HaCaT cells under hypoxia, so as to investigate the possible mechanism of CPT in the treatment of plaque psoriasis. Methods HaCaT cells were cultured respectively in normoxia and hypoxia condition for 12 hours, the CCL20 mRNA expressions were detected by real-time PCR, the secretions of CCL20 protein were detected by ELISA kit. HaCaT cells were divided into fi ve groups and treated with different nanomolar（12.5, 25, 50, 100, 200 nmol/L） of CPT, the fi ve groups were cultured in hypoxia condition for 12 hours, the secretions of CCL20 protein were detected by ELISA kit. Results There were signifi cantly difference in the expression levels of CCL20 m RNA（given in ΔCT value） between the normoxia group and the hypoxia group（-15.19±0.13 vs-13.70±0.10 t=-14.430, P=0.001）. The CCL20 protein secretion of HaCaT cells（1×105cells） were increased which cultured in hypoxia for 12 hours, however, there were no significantly difference in the CCL20 protein secretion of HaCaT cells（1×105cells） between the normoxia group and the hypoxia group [（112.18±28.66） pg/ml,（64.36±47.85）pg/ml, t=-1.485, P=0.212]. Hypoxia inducible for 12 hours, CPT of 100, 200 nmol/L signifi cantly decreased the secretion of CCL20 protein（1×105cells） compared with the vehicle control group [（64.35±19.70） pg/ml,（74.35±23.85） pg/ml vs（112.18±28.66）pg/ml, P〈0.05]. Conclusion The CCL20 mRNA expression of HaCaT cell is up-regulated in hypoxia, CPT of 100, 200 nmol/L can reduce the secretion of CCL20 protein.