热休克及化疗刺激对人白血病细胞K562中HSP70 mRNA及MDR1 mRNA表达的影响

为了研究热休克、Adriamycin（ADM）化疗应激刺激下热休克蛋白70（HSP70）、多药耐药基因MDRl的表达情况，对K562细胞予以43℃高温热休克和（或）加以ADM化疗，然后采用免疫组织化学方法检测HSP70的表达，MTT法检测K562细胞的生长抑制率，RT—PCR检测HSP70 mRNA和MDRl mRNA的表达，流式细胞术检测P-糖蛋白（P—gP）的表达。结果显示：①HSP70蛋白的合成在43℃热作用后恢复37℃孵育2小时达到高峰并聚集在胞核和核仁内，呈“核内迁移”现象；3小时后HSP70表达开始降低，5小时后HSP70的合成逐渐降低至正常，并恢复以胞浆表达为主。②K562细胞在43℃高温热休克刺激下HSP70mRNA表达增加，随着37℃孵育时间的延长表达量有增多趋势并持续一段时间，MDRlmRNA的表达与之相似，二者在37℃孵育2小时分别增加4倍和5．8倍。③ADM化疗刺激后细胞内P—gP表达增加；ADM化疗、43℃热休克刺激后再加ADM化疗，K562细胞中HSP70 mRNA、MDRl mRNA表达均上调，且43℃热休克刺激处理后再加入ADM作用二者的表达要高于单加ADM化疗组。结论：热休克和ADM化疗刺激的K562细胞中HSP70和MDRl表达增加，有利于维护肿瘤细胞的稳定性，HSP70可能与耐药有关。

Expression of HSP70 mRNA and MDR1 mRNA in K562 Cells Induced by Heat Shock and ADM

This study was purpose to investigate the expression levels of HSP70 and MDR1 genes under heat shock and/or adriamycin (ADM) chemotherapy stimulation. The K562 cells were bathed in water at 43 degrees C for 1 hour, then the heat-treated K562 cells were collected and were cultured at 37 degrees C. The expression of HSP70 was assayed by immunocytochemistry, the growth suppression rate of K562 cells was detected by MTT assay, the function of P-gp and the expressions of HSP70 mRNA, MDR1 mRNA were detected by flow cytometry and real-time quantitative PCR (RT-PCR) respectively. The results showed that (1) the synthesis of HSP70 protein in K562 cells treated with high shock (43 degrees C) reached to high level after culture at 37 degrees C for 2 hours, and moved from cytoplasm to nucleolus, the expression of HSP70 began to decrease following 3 hours of culture at 37 degrees C, and gradually reached to normal level after culture at 37 degrees C for 5 hours, the location of HSP70 expression returned to cytoplasm; (2) the expressions of HSP70 mRNA and MDR1 mRNA increased following 43 degrees C heat shock, and were 4 and 5.8 times higher than that of control group at 37 degrees C culture for 2 hours respectively; (3) the expression of P-gp was higher in ADM group than that in control. The expressions of HSP mRNA and MDR1 mRNA increased significantly in heat shock plus ADM group and ADM group as compared with control (p<0.01). It is concluded that the heat shock and ADM chemotherapy both induce over expression of HSP70 and MDR1 which can maintain stability of K562 cells and may be related to formation of the MDR in leukemia.