巨噬细胞炎症蛋白-1β对人舌鳞癌细胞CAL-27增殖和凋亡的影响

Effect of macrophage inflammatory protein-1β on proliferation and apoptosis of human tongue squamous cell carcinoma CAL-27 cells in vitro

目的探讨β亚族趋化因子受体5(CCR5)在不同人舌鳞癌细胞株的表达情况以及其配体巨噬细胞炎症蛋白-1β(MIP-1β)对人舌鳞癌细胞株CAL-27增殖和凋亡的影响。方法采用蛋白印迹法和免疫荧光染色对3种人舌鳞癌细胞株(CAL-27、UM-1、Tca-8113)MIP-1β的相应受体CCR5的表达进行检测;根据MIP-1β刺激浓度的不同,细胞被随机分为4组:0 ng/m L组(对照组)、10 ng/m L组、20 ng/m L组、40 ng/m L组。用CCK-8试剂盒(Cell Counting Kit-8)检测MIP-1β在刺激12、24、48 h时,对CAL-27细胞增殖的影响;实验分组同前,分别用0、10、20、40 ng/m L IP-10处理24 h后收集细胞,用Annexin V/PI双染流式细胞仪检测CAL-27细胞的凋亡情况。结果 CCR5在3种人舌鳞癌细胞株均有表达,并且CCR5在3株细胞的胞膜及胞质内均有染色;和对照组相比,MIP-1β对CAL-27细胞有促进增殖的作用。在12 h、24 h时,3种浓度的MIP-1β均能够促进CAL-27细胞增殖(P<0.05);48 h时,10 ng/m L、20 ng/m L的MIP-1β均能够促进CAL-27细胞增殖(P<0.05),但40 ng/m L的MIP-1β对CAL-27细胞的增殖无影响(P>0.05);在24 h时,40 ng/m L MIP-1β组的细胞凋亡率显著高于对照组(P<0.05),而10 ng/m L、20 ng/m L MIP-1β组细胞凋亡率和对照组之间均无显著差异(P>0.05)。结论 CCR5在3种人舌鳞癌细胞株的胞膜及胞质内均有表达;MIP-1β对CAL-27细胞有促进增殖的作用,但相对较高浓度的MIP-1β对CAL-27细胞的凋亡也有促进作用。随着较高浓度MIP-1β作用时间的延长,促增殖作用减弱,而这种减弱可能是由MIP-1β的促凋亡作用的逐步发挥所引起的。

Objective To detect CCR5 protein expression in different human tongue squamous cell carcinoma cells and observe the effect of macrophage inflammatory protein-1β （MIP-1β） on the proliferation and apoptosis of CAL-27 cells. Methods Western blotting and immunofluorescence staining were used to detect the expression of the CCR5, the receptor of MIP-1β, in 3 human tongue squamous cell carcinoma cells UM-1, CAL-27, and Tca-8113. CCK-8 assay was used to assess the proliferation of CAL-27 cells stimulated with 10, 20, and 40 ng/mL MIP-1β for 12, 24, or 48 h. The apoptosis of the cells stimulated with MIP-1β（10, 20, and 40 ng/mL） for 24 h was analyzed using flow cytometry with Annexin V/PI double staining. Results CCR5 expression was detected both on the membrane and in the cytoplasm in all the 3 tongue squamous cell carcinoma cell lines. At the concentrations of 10, 20, and 40 ng/mL, MIP-1β stimulation for 12 and 24 h significantly promoted the proliferation of CAL-27 cells （P〈0.05）;MIP-1βstimulation for 48 h at the concentrations 10 and 20 ng/mL, but not at 40 ng/mL, promoted the proliferation of CAL-27 cells （P〈0.05）. MIP-1βstimulation at 40 ng/mL for 24 produced the most obvious apoptosis-inducing effect in CAL-27 cells （P〈0.05）, while MIP-1β at 10 or 20 ng/mL did not induce obvious apoptosis in the cells （P〉0.05）. Conclusion CCR5 is expressed in all the 3 human tongue squamous cell carcinoma cells. MIP-1βcan promote the proliferation of CAL-27 cells but high concentrations of MIP-1βalso induced cell apoptosis. Prolonged stimulation of the cells with a high concentration of MIP-1βshows attenuated effect in promoting cell proliferation probably as a result of cell apoptosis induced by MIP-1β.