三氧化二砷对腺样囊性癌ACC-2细胞TNF-α基因表达的影响

The Effect of Arsenic Trioxide on the Expression of Tnf-α Gene in Adenoid Cystic Carcinoma Acc-2 Cells

摘要: 研究目的：本实验旨在通过研究As2O3对体外培养的ACC-2细胞增殖、凋亡的影响，从而探讨其对ACC-2细胞诱导凋亡的具体机制。研究方法：将ACC-2细胞进行体外培养并分为实验组和对照组，于细胞指数生长期向实验组加入不同浓度梯度(2.0、4.0、6.0、8.0 μmol/L)的含As2O3细胞培养液，对照组则给予等量全细胞培养液，加入As2O3后分别培养不同时间(12、24、36、48 h)，密切观察各时间点不同浓度As2O3对腺样囊性癌ACC-2细胞的生长抑制作用和细胞凋亡的形态变化，并应用荧光定量RT-PCR和免疫细胞化学技术检测TNF-α基因和蛋白的表达变化。研究结果：1) 随着加入As2O3浓度及时间增加，显微镜下可见胞体缩小变圆，细胞接触减少，细胞间隙变大，胞浆内颗粒样物质增多，胞核固缩，贴壁细胞减少，漂浮坏死的细胞逐渐增多。表示As2O3对ACC-2细胞的生长具有抑制作用，且随着药物浓度的升高，细胞的生长抑制作用逐渐加强。2) 免疫细胞化学检测结果示在细胞核和细胞浆均有TNF-α蛋白表达，以细胞核为主，不同浓度As2O3作用于ACC-2细胞后，阳性颗粒随浓度及作用时间增加逐渐向细胞核聚集并增强，测得的平均光密度值(IOD/area)逐渐增大，不同浓度总体比较TNF-α蛋白表达量差异有统计学意义，但不同处理时间总体比较TNF-α蛋白表达量差异无统计学意义；两两比较，不同浓度除0 μmol/L与2 μmol/L及6 μmol/L与8 μmol/L外其余均有统计学意义(P < 0.01)，不同时间除12 h与48 h外其余均无统计学差异(P > 0.05)。3) RT-PCR结果显示在空白对照组和实验组ACC-2细胞中TNF-α mRNA均存在表达，其表达量随着As2O3浓度的增加和作用时间的延长略有下降，但不同浓度及时间点总体比较差异均无统计学意义(P > 0.05)，不同浓度两两比较均无统计学差异(P > 0.05)，不同时间两两比较均无统计学差异(P > 0.05)，这与免疫组化结果不同。结论：As2O3对体外培养的ACC-2细胞具有生长抑制和诱导凋亡作用，能够上调腺样囊性癌ACC-2细胞中TNF-α蛋白的表达及分泌，使TNF-α在蛋白水平参与As2O3诱导ACC-2细胞的凋亡。

Abstract: Objective: The purpose of this study was to investigate the effect of As2O3 on the proliferation and apoptosis of ACC-2 cells cultured in vitro to detect its specific mechanism of ACC-2 cell apoptosis. Methods: ACC-2 cells were cultured in vitro and divided into experimental group and control group with the same amount of cell culturing. In the exponential growth phase the ACC-2 cells cultured in experimental group was added with different concentration of As2O3 (2.0, 4.0, 6.0 and 8.0 μmol/L) and the control group gave equal amount of cell culture medium. After added As2O3, the cells were cultured at different times (12 h, 24 h, 36 h, 48 h). Then the morphological changes of growth inhibition and apoptosis of the ACC-2 cells at each point time in different concentrations of As2O3 were closely observed. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry test (IHCT) were used to detect the expression changes of TNF-α gene and protein. Results: 1) With the increase of As2O3 concentration and time, it could be seen that the cells became smaller and round, the cell contact was reduced and the cell gap became larger, intracellular particles increased, cell density increased accompanied by vacuolar degeneration, adherent cells decreased, floating necrosis of the cells gradually increased, which shown that As2O3 could inhibit the proliferation of ACC-2 cells, and with the increase of drug concentration and time, the growth inhibition of cells gradually strengthened. 2) Immunohistochemical results showed that TNF-α proteins were expressed in the nucleus and the cytoplasm in TNF-α cells in both the experimental group and the control group. TNF-α proteins were expressed mainly in the nucleus. Positive particles gradually gathered to the nucleus and AIO value was increased gradually as the concentration increased. The difference was statistically significant (P < 0.01). 3) The results of RT-PCR showed that the TNF-α mRNA expressed in ACC-2 cells in both the experimental group and the control group, and its expression decreased slightly with increasing concentrations of As2O3 and prolonging duration of action. However, it was not statistically significant between the concentration and time groups (P > 0.05); it was not statistically significant between the two comparisons at different concentrations (P > 0.05) or different time (P > 0.05), which was different from immunohistochemical results. Conclusion: As2O3 could inhibit the growth and induce the apoptosis of Acc-2 cells cultured in vitro and could increase the expression and secretion of TNF-α protein in Acc-2 cells of adenoid cystic carcinoma, that is, TNF-α is involved in apoptosis of acc-2 cells induced by As2O3 in the protein level.