NF-κB p65抑制与X射线诱导的人非霍奇金淋巴瘤细胞凋亡的研究

Inhibition of NF-κB enhanced X-ray irradiation induced apoptosis in human non-Hodgkin lymphoma

目的 探讨NF-κB p65对X射线诱导人非霍奇金淋巴瘤（non-Hodgkin lymphoma, NHL）细胞凋亡的作用及其调控机制。方法 以NF-κB p65抑制剂quinazoline（QNZ）处理人NHL细胞。将NHL细胞株Namalwa、Ramos和 Raji细胞分为空白对照组、单纯照射组（IR）和X射线＋QNZ实验组（IR＋QNZ）,采用Annexin-Ⅴ染色方法检测肿瘤细胞凋亡水平的变化,采用Western blot方法检测各细胞株中Survivin及凋亡相关蛋白Bax、Bcl-2和Cleaved Caspase-3的表达水平;应用实时定量PCR方法检测Survivin mRNA水平。结果 应用流式细胞仪检测凋亡细胞百分比,结果显示,QNZ处理后进行照射与单纯照射组相比凋亡细胞明显增加,且具有药物浓度依赖性（t=2.93~12.52, P〈0.05）,Western blot法检测结果显示,电离辐射可显著增加人NHL细胞中Survivin蛋白的表达。而应用1、10和50 nmol/L QNZ处理人NHL细胞24 h后进行照射,可显著下调电离辐射所诱导的NHL细胞中的Survivin蛋白表达。随药物浓度增加其作用更为明显（t=3.29~16.72,P〈0.05）。同时凋亡相关蛋白Bcl-2表达减低,而Bax和Cleaved caspase-3蛋白表达增加,Bcl-2/Bax比值明显降低,与单纯照射组相比,差异有统计学意义（t=6.20~9.91, P〈0.05）。预处理QNZ后射线诱导的3种NHL 细胞Survivin mRNA均不同程度下降。结论 抑制NF-κB能够增加X射线诱导的NHL细胞凋亡,其机制可能与下调Survivin蛋白表达水平及对凋亡相关蛋白Bcl-2家族的调节有关。

Objective To investigate the role of NF-κB in radiation-induced apoptosis of human non-Hodgkin lymphoma （NHL） cells and the mechanism involved. Methods Three human non-Hodgkin lymphoma cell lines, Namalwa,Ramos, and Raji cells were divided into control, IR and IR＋QNZ （10 nmol/L） groups,respectively. Annexin-Ⅴ kit was used to determine cell apoptosis. Protein expression levels of Survivin, Bax, Bcl-2 and cleaved Caspase-3 were evaluated by Western blot. Survivin mRNA was quantified by real-time PCR. Results Inhibition of NF-κB by QNZ pretreatment significantly enhanced X-ray induced apoptosis in human NHL cells in a dose-dependent manner （t=2.93-12.52, P〈0.05）. At the same time, QNZ significantly reversed the expression levels of Survivin protein and mRNA that were up-regulated by radiation（t=3.29-16.72,P〈0.05）. QNZ also increased the levels of Caspase-3 and pro-apoptotic protein Bax, but reduced anti-apoptotic protein Bcl-2 expression level and hence the ratio of Bcl-2/Bax. Conclusions Inhibition of NF-κB could enhance radiation-induced cell apoptosis in human NHL cells through down-regulating Survivin expression and decreasing Bcl-2/Bax ratio.