毒胡萝卜素诱导K562细胞凋亡及其调控机制的研究

Study on thapsigargin-induced apoptosis and its regulatory mechanism in K562 cells

目的探讨毒胡萝卜素(TG)对K 562细胞凋亡诱导作用及其调控机制。方法Hoechst 33258染色荧光显微镜观察凋亡细胞形态改变;A nnex inⅤ-F ITC/P I双染检测细胞凋亡率变化;RT-PCR检测bcl-2、bax、bcl-XL、X IAP、surv iv in基因表达变化并通过免疫组织化学技术检测其在蛋白质水平的表达。结果TG作用后,K 562细胞呈典型的凋亡细胞形态;细胞凋亡率均明显升高(P<0.05),且在一定范围内呈浓度和时间依赖性;bax、bcl-XL、X IAP mRNA和蛋白质表达上调,surv iv in mRNA和蛋白质表达下调,bcl-2/bax比率降低。结论TG可诱导K 562细胞发生内质网应激反应性凋亡,其机制可能与B ax表达上调、surv iv in表达下调有关,bcl-XL、X IAP表达上调可能发挥拮抗凋亡的作用。

[Objective] To study the apoptotic induction effect of thapsigargin on leukemia cell lines K562 and its regulatory mechanism. [Methods] Morphological change of apoptotic cells was observed by Hoechst 33258 staining under fluorescent microscope, Apoptosis rate was determined with a Annexin V-FITC/PI double staining by flow cytometry. The expression of bcl-2, bax, bcl-XL,Survivin,XIAP genes mRNA and protein were detected respectively by RT-PCR and immunohistochemistry technology. [Results ] After exposure to thapsigargin, K562 cells exhibited typical morphological changes of apoptotic cell, apoptosis rate of K562 was higher than that of control group （P〈0.05）, and it was dose- and time-dependent in experiment range; Expression of bax, bcl-XL, XIAP genes mRNA and protein were up-regulated, survivin genes mRNA and protein were down-regulated, bcl-2/bax ratio decreased, [Conclusion] Thapsigargin can induce endoplasmic reticulum stress-induced apoptosis of K562 cells, its mechanismed may associate with up-regulation of bax expression and downregulation of survivin expresson, upregulation of bcl-XL and XIAP expression may play an important role against apoptosis.