作者：冯献启，游泳，肖娟，邹萍

【摘要】 本研究旨在探讨毒胡萝卜素对K562细胞的凋亡诱导作用及其可能机制。采用荧光显微镜观察凋亡细胞的形态变化，annexinⅤ-FITC/PI双染法FCM检测凋亡率的变化，Ca2 荧光指示剂Fura-2/AM法荧光分光光度计测定细胞内Ca2 浓度（〖Ca2 ］i）的改变，Rh123单染法FCM检测线粒体ΔΨm的变化，Western blot检测caspase-3，-7，-9，-12、细胞色素 C和GRP78蛋白的改变。结果显示：4 μmol/L 毒胡萝卜素作用K562细胞48小时后，荧光显微镜观察到细胞呈典型的凋亡形态变化，早期凋亡细胞核染色质呈固缩状、圆珠状或斑块状；晚期凋亡细胞核染色质呈固缩状或斑块状。1、2、4、8 μmol/L 毒胡萝卜素作用K562细胞24和 48小时后细胞凋亡率分别为7.51％、11.65％、23.22％、30.56％和12.85％、20.27％、31.51％、44.16％，在一定范围内呈剂量和时间依赖性，与对照组比较，均有统计学意义（P<0.05）。毒胡萝卜素诱导K562细胞〖Ca2 ］i升高以及线粒体ΔΨm下降，并均呈一定的浓度依赖性，与对照组比较，均有统计学意义（P<0.05）。Western blot检测显示，毒胡萝卜素诱导K562细胞caspase-3，-7，-9，-12剪切活化、细胞色素C释放、GRP78表达上调。结论:毒胡萝卜素可诱导K562细胞发生内质网应激反应性凋亡，内质网是细胞内诱导凋亡的一个重要新场所；caspase-3，-7，-9，-12剪切和活化、线粒体ΔΨm破坏和细胞色素C释放是毒胡萝卜素诱导K562细胞凋亡的重要机制之一，线粒体参与内质网应激反应性凋亡途径并发挥重要作用。

【关键词】 毒胡萝卜素

　　Thapsigargin-induced apoptosis of K562 cells and its mechanism

　　Abstract The aim was to study the apoptotic induction effect of thapsigargin on leukemia cell line K562 and its possible mechanism. After the treatment of leukemia cell line K562 by thapsigargin，morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope；apoptosis rate was determined with annexinⅤ-FITC/PI double staining by flow cytometry；intracellular calcium concentrations (〖Ca2 ］i) were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM；mitochondrial transmembrance potentials (ΔΨm) was detected on flow cytometry through staining of Rhodamine (Rh123)；the changes of caspase-3，-7，-9，-12、cytochrome C、GRP78 proteins were detected by Western blot. The results showed that K562 cells cultured in 4 μmol/L thapsigargin for 48 hours exhibited typical morphological changes of apoptotic cells under fluorescent microscope，including shrinkage of cell，condensation of chromatin，breakage of nuclear，formation of apoptotic bodies，fluorescence of yellow green and pellet observed in early apoptoyic cells and hyacinth fluorescence of chromatin showed in late apoptotic cells. 24 and 48 hours after exposure to 1，2，4，8 μmol/L thapsigargin，the apoptotic rates of K562 were respectively 7.51%，11.65%，23.22%，30.56% and 12.85%，20.27%，31.51%，44.16%，in dose- dependent manner，and were statistically significant when compared with the controls (P<0.05). The apoptotic rate of K562 was dose- and time-dependent in experiment range. The enhancement of 〖Ca2 ］i and the decrease of the ΔΨm in K562 cells were induced by thapsigargin and were dose-depend ent in experiment range，compared with control，P<0.05. Western blot results indicated that cleavage and activation of caspase-3，-7，-9，-12，releasing of cytochrome C from mitochondria，upregulation of GRP78 expression at the endoplasmic reticulum were induced in K562 cells after 24 hours exposure of 4 μmol/L thapsigargin. It is concluded that thapsigargin induces endoplasmic reticulum stress-induced apoptosis in K562 cells. Endoplasmic reticulum is a novel important initiatory site of apoptosis in cells；the cleavage and activation of caspase-3，-7， -9，-12 play very important role in endoplasmic reticulum stress-induced apoptosis of K562 cells and is one of the important mechanisms for thapsigargin-induced apoptosis. Thapsigargin-induced apoptosis in K562 cells is

associated closely with the disruption of the ΔΨm and the release of cytochrome C from mitochondria，mitochondria participates in endoplasmic reticulum stress-induced apoptosis in K562 cells.

　　Key words thapsigargin；leukemia cell line；K562 cell；apoptosis；endoplasmic reticulum；caspase

　　化疗是目前治疗白血病的主要手段。虽然造血干细胞移植术是治疗白血病的理想方法，但是大部分患者由于无合适供者或不适合移植最终因化疗无效而死亡。白血病化疗无效的主要原因是白血病细胞突变及原发或继发耐药。因此，临床上亟需寻找针对白血病细胞内不同靶位点的新疗法。最近研究发现，内质网可能是细胞内诱导凋亡的一个新场所，即内质网应激反应性凋亡途径，它不同于经典的凋亡途径：死亡受体途径和线粒体途径［1，2］。作为一种新的凋亡通路，其确切机制尚未完全清楚。本研究以白血病细胞系K562细胞为研究对象，以内质网Ca2 -ATP酶抑制剂毒胡萝卜素（thapsigargin，TG）为内质网应激诱导剂，初步探讨TG对K562细胞的凋亡诱导作用及其可能机制，为开辟白血病化疗新方案提供一定的实验和理论依据。

　　材料和方法

　　试剂和细胞系
　
　　细胞系K562细胞为本室常规培养；小牛血清、RPMI 1640培养液均购自Gibco公司；吖啶橙（AO）、溴化乙锭（EB）为Sigma公司产品；兔抗人caspase-3，-12，GRP78多克隆抗体、 鼠抗人caspase-7，-9，细胞色素C单克隆抗体、毒胡萝卜素、Ca2 荧光指示剂Fura-2/AM、化学发光剂LumiGLO、annexinⅤ-FITC试剂盒均购自晶美生物工程有限公司；考马斯亮兰CBBG250蛋白测定试剂盒购自南京建成生物工程研究所；荧光染料Rh123、Western blot常规试剂均购自北京中山生物技术有限公司。

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