HIF-1α在食管上皮细胞中的诱导表达及对光动力疗法的影响

Inducible expression of HIF-1α in esophageal epithelia cells and its effect on photodynamic therapy

目的:探讨食管上皮Het1A细胞经诱导表达HIF1α后对光动力学效应的影响。方法:HIF1α的稳定高表达采用氯化钴(CoCl2)化学诱导法,并应用光敏剂5ALA处理细胞,光动力疗法(photodynamictherapy,PDT)光照射采用波长>600nm的红光光源,细胞存活活性以MTS法测定,应用Westernblot测定HIF1α蛋白质水平,RNA干扰采用脂质体转染法,并利用TdT流式细胞法测定细胞凋亡率。结果:Het1A细胞经100μmol/L的CoCl2孵育4h后可诱导HIF1α蛋白的高表达;PDT后高表达HIF1α的细胞具有较高的细胞存活活性,且细胞凋亡率由未诱导时的16%降低至4%;不同浓度CoCl2诱导的HIF1α蛋白质水平与PDT后细胞存活活性相一致;siRNA转染阻断HIF1α的诱导表达,细胞又恢复了对PDT的应答能力。结论:HIF1α在Het1A细胞中的高表达使PDT效果下降;HIF1α部分通过抑制PDT引起的细胞凋亡而抵御PDT效应。提示HIF1α可作为治疗靶基因,降低肿瘤组织HIF1α表达水平可能有助于改善PDT临床疗效。

Objective： To study the effect of over expression of hypoxia inducible factor-1 alpha （HIF 1α） in esophageal epi thelial Her-1 A cells on the efficiency of photodynamic therapy （PDT）. Methods：Stable over expression of HIF 1α in Her-1 A cells was induced by incubation with cobalt chloride（CoCl2 ）. Cells were treated with photosensitizer 5-ALA followed by irradiation with visible red light at a wavelength above 600 nm. HIF 1α protein level was determined by western blot. The cell viability was tested by 3-（4,5-carboxymethoxyphenyl）-2-（4 sulfophenyl）-2H tetrazolium（MTS） methods, siRNA was transfected into Het1A cells by liposome mediation to knock down the HIF 1α expression. The apoptotic cell fraction was measured by TdT staining. Results：The over expression of HIF 1α can be induced by 4 h incubation with COC12 100 μmol/L in Het-1A cells. Het-1A cells over expressed HHIF 1α had a lower sensitivity to PDT than those without HIF 1α over expression. TdT staining showed that cell apoptotic rate decreased from 16% to 4% after incubation with CoCl2. This could be further evidenced by the result that various concentrations of CoCl2 could induce different levels of HIF1α protein, which was in accordance with the difference in cell viability after PDT. However, when the over expression of HIF 1α was knocked down by siRNA transfection, the sensitivity of the cells to PDT was recovered. Conclusion： Over expression of HIF 1α make Het-1A cells resistant to PDT. The attenuated PDT efficacy was achieved, at least in part, by inhibition of apoptosis. It suggested that decreasing the expression level of HIF 1α will help to improve the efficacy of PDT.