Ad．RGD—ING4对人鼻咽癌细胞CNE抑癌增效及其机制的研究

目的：研究带RGD的腺病毒介导的ING4对人鼻咽癌细胞CNE生长、凋亡及细胞周期的影响并探讨其可能调节机制。方法：以Ad．RGD-ING4及腺病毒空载体感染CNE细胞，RT—PCR法检测ING4基因的表达水平，Westernblot法检测目的蛋白的表达。用MTT试验检测ING4对CNE细胞生长情况的影响，用AnnexinV—PE／7一AAD双染色测定ING4对CNE细胞凋亡的影响，用PI单染色检测ING4对CNE细胞细胞周期的影响。RT—PCR检测p21、Bcl-2、Bax基因的表达水平的差异，Westernblot法检测Survivin蛋白、Cleaved—caspase3蛋白表达的差异。结果：CNE细胞感染Ad．RGD-ING472h后，ING4在CNE细胞中过表达，CNE细胞的生长受到明显抑制，细胞凋亡率明显升高，G2／M期出现明显阻滞。RT-PCR结果显示，感染Ad．RGD-ING4的CNE细胞中Bcl-2表达下降，p21、Bax表达升高，差异具有统计学意义（均P〈0．01）。Westernblot结果显示Survivin蛋白表达下降，Cleaved—caspase3蛋白表达升高。结论：Ad．RGD-ING4可以对鼻咽癌细胞株CNE的起到抑癌增效作用，这一作用可能是通过下调Bcl-2、Survivin表达及上调p21、Bax、caspase3实现的。

Synergistic tumor suppression by Ad. RGD-ING4 in human nasopharyngeal carcinoma cell CNE and its mechanism

Objective：To investigate the effect of adenovirus-mediated ING4 with RGD on proliferation , apoptosis and cell cycle of human nasopharyngeal carcinoma cell CNE and explore its probable mechanism. Method： CNE cells were infected with Ad. RGD-ING4 and adenovirus vector, ING4 gene expression level was detected by RT-PCR and the target protein expression was tested by Western blot. MTT assay was adopted to evaluate the elect of ING4 on cell growth of CNE, Annexin -V-PE/7-AAD Double staining was used to measure the elect of ING4 on apoptosis,and PI staining was used to measure the elect of ING4 on the cell cycle. Differential expression of P21, Bcl-2 and Bax gene was detected by RT-PCR,and Differential expression of Survivin and Caspase3 protein was detected by Western blot. Result：CNE cells were cultured with Ad. RGD-ING4 for 72 h , the results showed that ING4 was overexpressed in CNE cells ,the growth of CNE cells was obviously inhibited , apoptosis rate was significantly increased and G2/M phase was arrested apparently. The results of RT-PCR showed that Ad. RGD- ING4 significantly down-regulated the Bel-2 and up-regulates the Bax and P21 expression in CNE cells ,and the difference was statistically significant（P〈0. 01）. Western blot showed that the expression of Survivin was decreased and Cleaved-Caspase3 was increased. Conclusion： Ad. RGD-ING4 can play the role of tumor suppressor synergies on nasopharyngeal carcinoma cell CNE by down-regulating Bcl-2, Survivin expression and up-regulating P21, Bax and Cleaved-Caspase3 expression .