蛇毒心脏毒素诱导肺癌A549细胞的凋亡机制研究  

Study on the Mechanism of Snake Venom Cardiotoxin(CTX)Induced A549 Cell Apoptosis

作　　者：李凤君[1] 袁进[1] 韩丽萍[1] 蒋琳兰[1] 李玉华[1] 

机构地区：[1]广州军区广州总医院药剂科,广州510010

出　　处：《中国药房》2014年第7期583-586,共4页China Pharmacy

基　　金：全军医学科学技术研究"十一五"计划课题项目(No.06MA125)

摘　　要：目的:研究眼镜蛇心脏毒素(CTX)诱导肺癌A549细胞凋亡的机制。方法:MTT法测定CTX体外细胞毒性作用;吉姆萨染色法观察CTX对A549细胞形态学的影响;线粒体膜电位(JC-1)法检测CTX诱导A549细胞后细胞JC-1的变化;流式细胞仪检测CTX对A549细胞凋亡的影响;Western blot法测定细胞色素C、凋亡蛋白酶前体(Procaspase)-3、Procaspase-9的表达;通过S180小鼠肉瘤模型检测CTX体内抗肿瘤活性。结果:CTX作用于A549细胞的半数抑制浓度(IC50)为0.770μg/ml;0.5、1.0、2.0μg/ml CTX可引起A549细胞明显缩小,在高倍镜下可观察到胞核皱缩,形成染色质边集等形态学改变;流式细胞仪检测到CTX使A549细胞出现凋亡,并且具有量效关系;5μg/ml CTX作用于A549细胞0.5 h可使细胞JC-1降低;胞浆中检测到细胞色素C时,Procaspase-9的含量没有明显变化,随后逐渐减少,CTX诱导细胞凋亡具有时效关系;体内抑瘤实验显示在剂量为0.5、1.0、2.0 mg/kg时CTX对A549细胞有明显的抑制作用。结论:CTX对A549细胞有细胞毒性,可以使线粒体内细胞色素C释放,并激活Procaspase-9和Procaspase-3诱导A549细胞的凋亡。OBJECTIVE： To study the mechanism of snake venoms cardiotoxin （CTX） induced A549 cell apoptosis. METHODS： MTT was used to detect the in vitro cytotoxicity of CTX. Giemsa stain was employed to examine the effects of CTX on morphology of A549 cells. JC-1 assay was used to detect the change of mitochondrial membrane potential of A549 cells induced by CTX. Flow cytometry was used to detect the effects of CTX on the apoptosis of A549 cells. The expressions of cytochrome C, procaspase-3 and procaspase-9 in the cytosol fraction were analyzed by Western blotting. S180 mouse sarcoma model was used to check in vivo anti-tumor activity of CTX. RESULTS： IC50 of CTX to A549 cells was 0.770 μg/ml. CTX 0.5,1.0,2.0 μg/ml could induce morphological changes, such as condensed chromatin and chromatin margination in A549 cells. Flow cytometry showed CTX induced the apoptosis of A549 cells in a dose-dependent. The mitochondrial membrance potential of A549 cells was reduced at 0.5 h （CTX 5 μg/ml） manner. The Procaspase-9 was not activated initially, then grodually reduced, whereas the cleavage of Cytochrome C was detected. The apoptosis of cells was induced in pragmatic relation. Inhibitory rates of CTX 0.5, 1.0 and 2.0 mg/kg on S180 cell tumor were inhibition respectively. CONCLUSIONS： CTX can significantly inhibit the growth of A549 cells. Once cytochrome C release from the mitochondria, cytochrome C activate Procaspase-9 and Procaspase-3 to leading to the apoptosis.

关 键 词：A549细胞 眼镜蛇蛇毒 心脏毒素 凋亡 

分 类 号：R285[医药卫生—中药学] R549[医药卫生—中医学]