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目的:探讨NF-κB信号转导通路在放射线诱导的人唾液腺腺样囊性癌(ACC)-2细胞凋亡中的变化规律。方法:采用脂质体2000将pBabe-SR-IκBa质粒瞬时转染入ACC-2细胞,Western blot法检测转染后IκBa蛋白表达的变化,将转染后的ACC-2细胞进行6种剂量(0、2、4、6、8、10 Gy)的高能X线照射,在照射后不同时间点(1、3、6、10、24、48 h)应用免疫细胞化学检测ACC-2细胞NF-κB p65的表达情况,应用Image Pro-Plus图像分析软件测其细胞核平均灰度值,并用SPSS 16.0软件对数据进行方差分析。结果:与转染pBabe空白质粒组和未转染质粒组比较,转染pBabe-SR-IκBa质粒ACC-2细胞不仅表达内源性IκBa,同时也表达SR-IκBa;接受相同放射线剂量照射后3 h细胞核平均灰度值升高(P<0.05)。SR-IκBa质粒组中,不同剂量组的细胞核平均灰度值均低于0 Gy组;约在接受照射后6～10 h,细胞核平均灰度值达到最低值;相同时间点上,3 h组细胞细胞核平均灰度值随放射剂量增加而降低(P<0.05)。结论:转染突变的IκBa基因可特异性抑制NF-κB信号通路,经放射线照射诱导凋亡后,NF-κB p65蛋白的表达量具有一定的时间-剂量效应规律。
Objective: To investigate the changes of NF-κB signal transduction pathway in radiation-induced apoptosis of human salivary adenoid cystic carcinoma (ACC) -2 cells. Methods: The plasmid pBabe-SR-IκBa was transiently transfected into ACC-2 cells by lipofectamine 2000. The expression of IκBa protein was detected by Western blot. The transfection of ACC-2 cells was performed in 6 doses 0, 2, 4, 6, 8 and 10 Gy). The immunocytochemistry was used to detect the expression of NF-κB in ACC-2 cells at different time points after irradiation (1,3,6,10,24,48 h) κB p65 expression using Image Pro-Plus image analysis software to measure the average gray value of the nucleus, and using SPSS 16.0 software analysis of variance data. RESULTS: ACC-2 cells transfected with pBabe-SR-IκBa plasmid not only expressed endogenous IκBa but also expressed SR-IκBa as compared with those transfected with pBabe blank plasmid group and untransfected plasmid group. After receiving the same dose of radiation dose 3 h, mean gray value of nucleus increased (P <0.05). In the SR-IκBa plasmid group, the average gray value of nuclei in different dose groups was lower than that in 0 Gy group; mean gray value of nuclei reached the lowest value at 6-10 hours after irradiation; at the same time point, The average gray value of cell nucleus decreased with the increase of radiation dose (P <0.05). CONCLUSION: Transfection of IκBa gene specifically inhibits NF-κB signaling pathway. After induced by radiation, the expression of NF-κB p65 protein has some time-dose effect.