缺氧促进舌苔形成相关细胞凋亡的分子机制

Molecular mechanism of hypoxia-induced apoptosis in tongue coating-related cells

目的探讨缺氧对舌苔形成相关细胞凋亡的影响。方法将Co Cl2作用于撤血清舌鳞癌Tca-8113细胞,建立化学性缺氧模型。MTT法检测细胞增殖活性,荧光染色检测细胞凋亡,琼脂糖电泳观察DNA Ladder,流式细胞术检测细胞周期分布和凋亡,Western blotting检测Akt、NF-κB p50、NF-κB p65、Bax、Bcl-2、HSP 70的表达水平。结果 50-400μmol/L Co Cl2作用细胞8-24h能明显抑制撤血清舌鳞癌细胞增殖（P〈0.05）,并具有量效和时效关系。Hoechst荧光染色显示Co Cl2可引起撤血清舌鳞癌细胞凋亡。流式细胞术显示100,200,400μmol/L Co Cl212h缺氧-12h复氧可促进撤血清舌鳞癌细胞凋亡（P〈0.05）,且凋亡率随Co Cl2的浓度增高。Western blotting表明12h缺氧-12h复氧可上调Bax、HSP 70、NF-κB p50,下调NF-κB P65表达（100μmol/L Co Cl2）,促进舌鳞癌细胞凋亡。结论缺氧是促进舌苔形成相关细胞凋亡的重要因素,其发生机制可能受NF-κB、Bax、HSP 70蛋白的调控。

Objective To investigate the hypoxia- induced apoptosis in tongue coating- related cells. Methods Cobalt chloride（ Co Cl2）,a chemical hypoxia inducer,was added in Tca- 8113 cells with serum withdrawal to develop a hypoxic model. Proliferative activity of Tca- 8113 cell was detected by MTT assay. Apoptosis were detected by Hoechst 33342 staining. DNA fragments in Tca- 8113 cells were analyzed by gel electrophoresis for visualizations of apoptosis- indicative DNA ladders. The distribution of cell cycle and apoptosis were detected by flow cytometric analysis. The expressions of Akt,natural factor- κB（ NF-κB） p50,NF- κB p65,Bax,Bcl- 2 and heat shock proteins（ HSP） 70 were detected by Western blot. Results Compared with control group,proliferative activity of Tca- 8113 cells decreased after Co Cl2（ 50 - 400μmol/L） acting for 8 hours（ P〈0. 05） in a dose- effect dependent and time- effect dependent manner. Hoechst 33342 staining showed that Co Cl2 promoted apoptotic rate of Tca- 8113 cells. Treated with Co Cl2（ 100,200,400μmol/L） in 12 h hypoxia- 12 h reoxygenation,apoptotic rate of Tca-8113 cells increased significantly（ P〈0. 05） in a dose- effect dependent manner. Western blot analysis showed that 12 h hypoxia- 12 h reoxygenation lead to increase of Bax,HSP70 and NF- κB p50（ 100μmol / L Co Cl2） but not Akt and Bcl- 2,and decrease of NF- κB p65（ 100μmol / L Co Cl2）. Conclusion Hypoxia is an important factor in the apoptosis of tongue coating- related cells. And its mechanism may through regulation of Bax,HSP 70 and NF- κB.