HSP70/HSP90在鼻咽癌热疗后的表达变化及对热疗疗效影响的体外研究

In vitro Study of Expression and Effects of HSP70/HSP90 in Nasopharyngeal Carcinoma Cells after Thermotherapy

目的探讨体外鼻咽癌细胞系HNE1细胞热疗后热休克蛋白70/90(HSP70/HSP90)的表达变化及抑制HSP70/HSP90表达对鼻咽癌热疗的影响,并初步探讨其机制。方法对体外培养的鼻咽癌细胞HNE1,恒温水浴42℃热处理2h,分别于2h、4h、6h、8h、12h、24h后荧光定量PCR检测HSP70/HSP90的基因表达变化,采用Westernblot检测蛋白表达量的变化。流式细胞术检测细胞的凋亡,观察在热疗前应用槲皮素、格尔德霉素及联合应用对HSP70/HSP90表达抑制后,对HNE1细胞凋亡的影响。结果42℃热疗后鼻咽癌细胞HSP70/HSP90表达在基因及蛋白水平上均有明显升高,4h达到高峰,8h后开始回落,24h接近正常。槲皮素能抑制HSP70表达,但导致HSP90表达水平升高及下降延迟。热疗前使用槲皮素及格尔德霉素处理,凋亡细胞比例明显增加,联合处理组增加更为明显(P<0.05)。结论HSP70/HSP90在HNE1细胞内的表达均在热疗后迅速升高,在热疗前联合抑制HSP70/HSP90的活性,可以更有效增加热疗敏感性。

Objective To investigate the changes and effects of HSP70/HSP90 of in nasopharyngeal carcinoma cells HNE1 after thermotherapy. Methods HNE1 cells were incubated at 42℃ for 2 h. The changes of mRNA and protein level of HSP70/HSP90 were detected by real-time PCR and western-blot at different intervals. HNE1 cells were pretreated with quercetin, geldanamycin or quercetin plus geldanamycin, respectively, before heat treatment. Flow cytometry assay was applied to determine the apoptosis of HNE1 cells before thermotherapy and at 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after thermotherapy. Results HSP70/HSP90 expression was up regulated at 2 h, reached to its peak at 4 h, descended at 8 h and returned to the normal level at 24 h after thermotherapy. The inhibition of HSP70 through quercetin induced up-regulation and delayed descent of HSP90. Combined pretreatment with quercetin and geldanamycin could significantly induce HNE1 apoptosis when compared with pretreatment with quercetin or geldanamycin alone （P 〈 0. 05）. Conclusion HSP70/HSP90 expression in HNE1 up regulated promptly after thermotherapy. Inhibition of expression and activity of HSP70/HSP90 before thermotherapy can increase sensitivity of the tumor cell to heat treatment.