Survivin 抑制剂 YM155对视网膜母细胞瘤Y79细胞线粒体凋亡途径的影响

摘要

目的：探讨人生存素（survivin）抑制剂YM155｛4，9－二氢－1－（2－甲氧基乙基）－2－甲基－4，9－二氧代－3－（2－吡嗪甲基）－1H－萘并[2，3－d]咪唑鎓溴化物｝对视网膜母细胞瘤Y79细胞凋亡、线粒体膜电位（Δψm ）和细胞色素C（Cyt C）的影响，探讨其诱导Y79细胞凋亡的线粒体机制。方法：体外培养Y79细胞，分别以0、0．5、1、2、4、8 nmol／L的YM155进行处理，采用CCK－8法和溴脱氧尿嘧啶核苷（ bromodeoxyuridine ，BrdU）掺入标记法检测YM155对Y79细胞增殖的抑制作用。 Y79细胞随机分为4组：对照组、阳性对照组[加入10 nmol／L拓扑替康（ topotecan ）]和低剂量（1 nmol／L）、高剂量（2 nmol／L） YM155组。每组设3个复孔，处理24 h。分别采用TUNEL染色和Annex－in V－FITC／PI双染法检测凋亡的情况；采用JC－1活细胞染色检测Δψm 的变化；采用免疫荧光分析检测Cyt C的分布；采用Western blot法检测survivin和线粒体内Cyt C的蛋白表达。结果：与对照组比较，YM155对Y79细胞增殖具有明显抑制作用，并诱导Y79细胞凋亡（P＜0．05）；YM155显著降低Y79细胞的Δψm，促进Cyt C由线粒体释放至胞浆，降低线粒体内Cyt C的水平（ P＜0．05）。结论： YM155对Y79细胞增殖具有抑制作用，并诱导细胞凋亡，其机制可能是通过线粒体介导的细胞凋亡途径实现的。%AIM:To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mito-chondrial membrane potential (Δψm) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochon-drial mechanisms of apoptosis .METHODS:Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L.The cells in control group were treated without YM 155.The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine ( BrdU) labeling assay .Y79 cells were randomly divided into 4 groups:control group ( with equal volume of RPMI-1640 nutrient medium ) , positive control group ( 10 nmol/L topotecan ) , low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group.The effects of YM155 on the apoptosis, the changes of Δψm , the mitochondrial distribution and the protein level of Cyt C in the Y 79 cells were evaluated by flow cytom-etry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot , respectively.RE-SULTS:Compared with control group , YM155 significantly inhibited the proliferation of Y 79 cells and induced apoptosis (P<0.05).YM155 significantly reduced Δψm of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05).CONCLUSION:YM155 inhibits Y79 cell proliferation and induces apoptosis , and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway .