【作者】 王亚利； 王中卫； 王西京； 金迎迎；

【Author】 WANG Ya-li,WANG Zhong-wei,WANG Xi-jing,JIN Ying-ying Department of Oncology,The 2nd Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China

【机构】 西安交通大学附属第二医院；

【摘要】 目的探讨X线诱导后鼻咽癌CNE-2细胞凋亡存在形式以及Caspase-3酶活性、mRNA、蛋白的表达及相互之间的关系,拟阐明Caspase-3在X线诱导的鼻咽癌细胞凋亡中的调控及意义。方法用不同剂量X线照射鼻咽癌CNE-2细胞,于不同时间检测细胞形态学及早期细胞凋亡率变化,分别用比色法、Western blot、RT-PCR检测Caspase-3酶活性、蛋白含量、mRNA,同时设立酶活性抑制剂AC-DEVD-CHO对照组,观察Caspase-3酶活性被阻抑后细胞凋亡及Caspase-3表达的变化。结果 (1)X线照射后鼻咽癌CNE-2细胞呈典型的凋亡特征,且早期凋亡率与射线剂量、诱导时间成正比;(2)CNE-2凋亡细胞中Caspase-3酶活性明显增高,用AC-DEVD-CHO抑制Caspase-3活性后细胞的凋亡率减少,且凋亡形态学特征不明显。(3)凋亡细胞中Caspase-3 mRNA表达量未见明显升高,而蛋白在凋亡形成后则完全裂解为17kD的活性Caspase-3,在正常细胞为32kD的pro-Caspase-3。结论 X线诱导后CNE-2细胞呈典型的凋亡改变。凋亡细胞中有明显的Caspase-3激活,其Caspase-3的活性增高并非源于其转录的提高,而可能是翻译后酶前体的活化。抑制鼻咽癌Caspase-3酶活性,凋亡的发生可被抑制或是延迟。更多还原

【Abstract】 Objective To identify and illuminate the effect and the mechanism of Caspase-3 gene of media-ting X radiation induced apoptosis in Nasopharyngeal carcinoma(NPC).Methods The CNE-2 cells wereexposed in X-radiation with different doses and incubated for different hours. Apoptosis were measured using Electron Microscopy(ECM) for morphology and Flow Cytometry (FCM) analysis with Annexin -Ⅴ/ PI dual staining for apoptotic cell ratio.The Caspase-3 mRNA and protein expression and enzyme ac-tivity in CNE-2 cells were investigated with RT-PCR,Western blot and colorimetric assay respectively.Results The predominant death model of NPC cells irradiated by X radiation caused apoptosis,and the apoptotic ratio was at dose and time dependent patterns;the Caspase-3 enzyme activity was significantly increased in apoptotic cells.With AC-DEVD-CHO inhibiting the enzyme activity,the apoptotic ratio of CNE-2 cells was decrease markedly(P < 0.05) and the character of apoptosis was not typical.The ex-pression of Caspase-3 mRNA in apoptotic CNE-2 cells was not significantly increased.The expression of protein was17kd active-Caspase-3 in apoptotic CNE-2 cells,while the 32kD pro-Caspase-3 was dominant in cont rol CNE-2 cells.Conclusion CNE-2 cells appeared characteristic apoptosis after X radiation.The activity of Caspase-3 plays an important role in apoptosis.The elevation of Caspase-3 activity in CNE-2ceIl may due to pro-caspase-3 being activated,not at transcription level.Apoptosis could be rest rained or retarded after the activation of Caspase-3 being inhibited.更多还原