肿瘤坏死因子对喉鳞状细胞癌细胞的增殖抑制作用

Inhibitory effect of tumor necrosis factor on proliferation of laryngeal squamous cell carcinoma

目的探讨肿瘤坏死因子(TNF)对喉鳞状细胞癌细胞的增殖抑制作用。方法选用人喉鳞癌Hep-2细胞株,分为对照组、20 pg/mL TNF-α组、50 pg/mL TNF-α组、100 pg/mL TNF-α组和200 pg/mL TNF-α组,分别采用相应药物进行处理。采用MTT法检测细胞增殖抑制率,采用流式细胞术检测细胞凋亡率和细胞周期,采用免疫组织化学法检测细胞增殖细胞核抗原(PCNA)表达水平,采用Western blot法检测细胞周期蛋白依赖性激酶4(CDK4)表达水平。结果与对照组比较,各浓度TNF-α组Hep-2细胞增殖抑制率及凋亡率较高,且20 pg/mL TNF-α组<50 pg/mL TNF-α组<100 pg/mL TNF-α组和200 pg/mL TNF-α组;与24 h比较,相同浓度TNF-α作用48 h后细胞增殖抑制率及凋亡率较高,差异有统计学意义(P<0.05);与对照组比较,各浓度TNF-α组Hep-2细胞S期细胞比例较低,G2/M期细胞比例较高;且在20~100 pg/mL浓度范围内,TNF-α浓度越高,G2/M期细胞阻滞越严重(P<0.05);与对照组比较,各浓度TNF-α组Hep-2细胞PCNA表达水平及CDK4活性较低;且20 pg/mL TNF-α组>50 pg/mL TNF-α组>100 pg/mL TNF-α组和200 pg/mL TNF-α组;与24 h比较,相同浓度TNF-α作用48 h后Hep-2细胞PCNA表达水平及CDK4活性较低,差异均有统计学意义(P<0.05)。结论 TNF对喉鳞状细胞癌Hep-2细胞增殖具有抑制作用,其能够使细胞阻滞于G2期,促进细胞凋亡,其作用可能与下调PCNA表达水平和CDK4活性有关。

Objective To analyze the inhibitory effect of tumor necrosis factor（TNF） on proliferation of laryngeal squamous cell carcinoma.Methods Human laryngeal squamous cell carcinoma Hep-2 cell were selected and divided into control group,20 pg/mL TNF-αgroup,50 pg/mL TNF-αgroup,100 pg/mL TNF-αgroup,200 pg/mL TNF-αgroup.Cells in each group were treated with the corresponding drugs.The cell proliferation inhibition rate was detected by MTT method,and flow cytometry was used to detect apoptosis rate and cell cycle.The expression of proliferating cell nuclear antigen（PCNA） was detected by immunohistochemistry,and the expression levels of cyclin dependent kinase 4（CDK4） were detected by Western blot.Results Compared with control group,the proliferation inhibition rate and apoptosis rate of Hep-2 cell were higher in different TNF-α concentration groups,with 20 pg/mL TNF-αgroup50 pg/mL TNF-α group100 pg/mL TNF-αgroup and 200 pg/mL TNF-αgroup.Compared with 24 h,the proliferation inhibition rate and apoptosis rate after 48 h treatment with the same concentration of TNF-α were higher,and the difference was statistically significant（P0.05）.（2） Compared with control group,the percentage of S cells in Hep-2 cells of different TNF-α concentration group were lower,and the proportion of G2/M cells were higher.In the 20-100 pg/mL concentration range,the higher the concentration of TNF-α,the more serious the blockage of G2/M phase cell（P0.05）.（3） Compared with control group,the expression level of PCNA and activity of CDK4 enzyme of Hep-2 cell were higher in different TNF-α concentration groups,with 20 pg/mL TNF-αgroup50 pg/mL TNF-α group100 pg/mL TNF-α group and 200 pg/mL TNF-αgroup.Compared with 24 h,the expression level of PCNA and activity of CDK4 enzyme after 48 h treatment with the same concentration of TNF-α were lower（P0.05）.Conclusion TNF can inhibit the proliferation of laryngeal squamous cell carcinoma cell line Hep-2,which can block the cells（in the G2 phase） and promote cell apoptosis.The effect may be related to down-regulation of PCNA expression and CDK4 enzyme activity.