三氧化二砷诱导鼻咽癌CNE1细胞凋亡及其作用机制的实验研究

Experimental Studies on Apoptosis in Human Nasopharyngeal Carcinoma CNE1Cell Line Induced by Arsenic Trioxide and Its Mechanism

目的研究三氧化二砷(As2O3)诱导人鼻咽癌细胞株凋亡作用及其相关机制。方法采用流式细胞术、电镜、TUNEL方法检测As2O3诱导CNE1细胞凋亡作用,免疫组织化学法检测As2O3对CNE1细胞p53、bcl-2和bax蛋白表达的影响。结果经As2O3处理的CNE1细胞发生凋亡,表现为FCM可检测到“亚二倍体峰”,形态学上可见核固缩,染色质边集,凋亡小体形成等改变;TUNEL法可检测到细胞内呈棕色颗粒的凋亡细胞,随着药物浓度升高,凋亡细胞发生率逐渐增多,As2O3目浓度为0.5mg/L、1.0mg/L和2.0mg/L作用48h后,CNE1细胞的凋亡指数分别为2.66±0.64、8.15±0.96和11.59±0.68,显著高于非药物处理组(0.43±0.43,P<0.05)。经As2O3处理的CNE1细胞内p53和bax蛋白表达较对照组明显增加,与凋亡指数呈正相关关系(r=0.554,P=0.011;r=0.891,P=0.000…);p53和bax蛋白表达也呈正相关关系(r=0.626,P=0.003)。结论As2O3在体外可诱导鼻咽癌CNE1细胞凋亡,其机制可能与上调p53、bax基因表达有关。

Objective To investigate the apoptosis-inducing effect of arsenic trioxide （As2O3 ） on human nasopharyngeal carcinoma and its possible mechanism. Methods CNEI cell line was treated with As2O3 at different concentration. Cell apoptosis was evaluated by flow cytometry, transmission electron microscopy and TUNEL methods. The effect of As,03 on the expression of p53, bax and bcl-2 genes was studied with immunohistochemology. Results CNEI cell apoptosis induced by As2O3 was detected by FCM, electron microscopy and TUNEL. Typical subdiploid peak before G0/G1 phase was observed by flow cytometric analysis, showing a dose-and time-dependent effect. Morphological feature of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation and formation of apoptotic bodies were found under electron microscopy. After 48h exposure to As2 03 at dose of 0. 5mg/L, 1.0mg/L and 2.0mg/L apoptosis index （AI） accounted by TUNEL staining were 2. 66 ± 0. 64,8. 15 ± 0. 96 and 11.59± 0. 68, respectively, which were significantly higher than that of control （0. 43 ± 0. 43,P〈0.05）. The expression of p53, bax protein was increased sharply in CNEI cell treated with As203. Significant positive correction existed between AI and p53 protein expression （r = 0. 554, P = 0. 011 ）, AI and bax protein expression （r = 0. 891, P = 0. 000…）. There was positive correction between p53 and bax protein expression （r = 0. 626, P = 0. 003）. Conclusion Arsenic trioxide can induce CNEI cell apoptosis, which was associated with up-regulation of bax and wild-type p53 genes expression.