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目的:将唾液腺腺样囊性癌ACC-2细胞在高能X线照射后,对细胞中NF-κB信号转导通路亚单位—p65蛋白表达及细胞凋亡情况进行检测,探讨两者的相关性。方法:将处于对数生长期的ACC-2细胞进行5种剂量(2、4、6、8、10Gy)的高能X线照射,照射后1、3、6、10t、24、48h,采用免疫细胞化学及免疫蛋白印迹(Western blotting)法,检测ACC-2中p65蛋白的表达情况;采用流式细胞仪测定细胞凋亡率,并进行TUNEL试验,观察凋亡细胞核形态;采用SPSS11.5软件包中的Spearman等级相关法进行放射线照射剂量强度与细胞凋亡率之间的分析。结果:通过免疫细胞化学观察,正常对照组细胞中p65蛋白多被定位于核周,而很少出现在胞核内;放射线照射后,该蛋白穿过胞膜,渐进入胞核中心;借助Western印迹法检测,p65蛋白在总蛋白中的含量可随时间逐渐变化,在各组细胞中表达峰值约在照射后6～10h;在同一时间点上,蛋白表达量随放射剂量的增加而增大。照射后,细胞凋亡率不断改变,其变化曲线最低点位于放疗后10h,并表现出对放射线剂量和p65蛋白含量的负相关关系(P=0.010<0.05);凋亡细胞在TUNEL试验中出现较典型的形态学变化。结论:放射线对ACC-2细胞p65蛋白表达的影响有一定的时间—剂量效应规律,细胞凋亡率与其表达量呈负相关。
OBJECTIVE: To detect the expression of NF-κB signaling pathway subunit-p65 protein and apoptosis in ACC-2 cells of salivary adenoid cystic carcinoma after high-energy X-ray irradiation, and to explore the correlation between them . Methods: ACC-2 cells in logarithmic growth phase were irradiated with high energy X-ray at 5 doses (2, 4, 6, 8 and 10 Gy). The mice were immunized with 1,3,6,10 and 24,48 h after irradiation Cytochemistry and Western blotting were used to detect the expression of p65 protein in ACC-2 cells. The apoptotic rates of ACC-2 cells were determined by flow cytometry. TUNEL assay was performed to observe the morphological changes of apoptotic nuclei. Spearman rank correlation method in the package was used to analyze the relationship between dose intensity of radiation and rate of apoptosis. Results: Immunocytochemistry showed that the p65 protein in the normal control group was mostly located in the perinuclear and rarely in the nucleus; after irradiation, the protein passed through the cell membrane, gradually into the nucleus center; by Western Western blotting showed that the content of p65 protein in total protein changed gradually with time, and the peak value of expression of p65 protein was about 6-10 hours after irradiation. At the same time point, the protein expression increased with the increase of radioactive dose . After irradiation, the rate of apoptosis changed constantly, and the lowest point of its change curve was at 10h after radiotherapy, and showed a negative correlation with radiation dosage and p65 protein content (P = 0.010 <0.05). Apoptotic cells appeared in TUNEL test More typical morphological changes. CONCLUSION: The effect of radiation on the expression of p65 protein in ACC-2 cells has some time-dose effects and the rate of apoptosis is negatively correlated with the expression of p65 protein.