Caspase-3在X线诱导的鼻咽癌细胞凋亡中的

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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-Casoase-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.“,”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 were exposed 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 apoptotie 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 control 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-2 cell may due to pro-caspase-3 being activated, not at transcription level Apoptosis could be restrained or retarded after the activation of Caspase-3 being inhibited.
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