双酚A对甲状腺乳头状癌细胞KTC-1增殖的影响
Effects of bisphenol A on proliferation of thyroid papillary carcinoma KTC-1 cells
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摘要:
背景 双酚A（BPA）是一种常见的环境内分泌干扰物，其化学结构与雌激素类似，近年来其与肿瘤之间的关系引起普遍关注。甲状腺癌是内分泌系统中发病率最高的恶性肿瘤，BPA对其发生发展的影响值得进一步探索。
目的 研究BPA对人甲状腺乳头状癌细胞增殖的影响及相关机制。
方法 体外培养人甲状腺乳头状癌细胞KTC-1后传代，随机分为对照组、BPA处理组（BPA浓度分别为5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8、5×10-9 mol·L-1），染毒24、48、72 h后，细胞增殖实验（CCK-8）检测细胞增殖活性，倒置显微镜观察细胞形态学改变，流式细胞仪分析细胞周期，免疫印迹法分别检测细胞周期蛋白（Cyclin）D1和细胞周期蛋白依赖激酶抑制剂（P21）的表达水平。
结果 5×10-3 mol·L-1 BPA诱导细胞大量死亡并漂浮于细胞培养液中，残留贴壁细胞出现较多空泡颗粒，细胞间连接消失，且时间越长形态学改变越明显；5×10-7、5×10-9 mol·L-1 BPA处理组细胞呈梭形，同时可观察到明显的细胞分裂现象。与对照组相比，5×10-3、5×10-4、5×10-5 mol·L-1 BPA诱导细胞不同时间（24、48、72 h）的细胞增殖率均有下降（
P < 0.05）；5×10-7、5×10-8、5×10-9 mol·L-1 BPA诱导细胞48 h增殖率较对照组明显升高（P < 0.001）；5×10-6 mol·L-1 BPA随着染毒时间的增加细胞增殖率变化不同，在处理24 h后，较对照组下降（P < 0.05），但在处理48、72h后明显升高（P < 0.001）。5×10-5、5×10-6、5×10-7、5×10-8、5×10-9 mol·L-1 BPA处理细胞24 h后流式细胞仪分析结果显示：除5×10-5 mol·L-1 BPA处理组，5×10-6~5×10-9 mol·L-1浓度组的细胞增殖指数（PI ）与对照组相比呈现上升趋势，但差异未见统计学意义；但其中5×10-6 mol·L-1 BPA处理组KTC-1细胞进入DNA合成期（S期）的细胞构成比高于对照组（P < 0.05）。与对照组相比，5×10-5 mol·L-1 BPA处理组Cyclin D1蛋白表达水平下降，P21蛋白表达水平上升；5×10-8、5×10-9 mol·L-1浓度处理组Cyclin D1蛋白表达水平上调，P21蛋白表达水平下降，差异均有统计学意义（P < 0.05）。结论 BPA对人甲状腺乳头状癌细胞KTC-1的增殖具有双重效应：高剂量BPA对甲状腺乳头状癌细胞KTC-1有毒作用，导致细胞死亡；低剂量BPA对甲状腺乳头状癌细胞KTC-1有促增殖作用。不同浓度BPA可能通过调节细胞周期蛋白Cyclin D1和P21的蛋白表达水平来促进或者抑制甲状腺乳头状癌细胞KTC-1的增殖。
Abstract:Background Bisphenol A (BPA) is a common environmental endocrine disruptor with a similar chemical structure to estrogen. In recent years, the relationship between BPA and tumors has attracted widespread attention. The effects of BPA on the development of thyroid cancer, with the highest incidence rate among endocrine system cancers, are worth exploring further.
Objective This experiment investigates the effects and mechanisms of BPA on the proliferation of human papillary thyroid carcinoma cells.
Methods Human papillary thyroid carcinoma KTC-1 cells were cultured
in vitro and passaged before the cells were randomly divided into two groups:control group and BPA experiment group (subgrouping by BPA concentrations at 5×10-3, 5×10-4, 5×10-5, 5×10-6, 5×10-7, 5×10-8, and 5×10-9 mol·L-1). After 24 h, 48 h, and 72 h exposure, CCK-8 experiment was used to detect the cell proliferation rate, inverted microscope was used to observe the morphological changes of cells, flow cytometry was used to analyze cell cycle, and Western blotting was used to detect the expression levels of Cyclin D1 and P21.Results BPA at 5×10-3 mol·L-1 induced massive cell death as the cells were floated in the medium, residual adherent cells were vacuolated, cell connection disappeared, and the morphological changes were more obvious with extended exposure time. The cells treated with BPA at 5×10-7 and 5×10-9 mol·L-1 were spindle-shaped, and significant cell division was observed. Compared with the control group, BPA at 5×10-3, 5×10-4, and 5×10-5 mol·L-1 significantly reduced the cell proliferation rate at different time points (24 h, 48 h, and 72 h) (
P < 0.05); BPA at 5×10-7, 5×10-8, and 5×10-9 mol·L-1 increased the cell proliferation rate at 48 h (P < 0.001); BPA at 5×10-6 mol·L-1 decreased the cell proliferation rate at 24 h, and increased the rate at 48 h and 72 h (P < 0.001). The results of flow cytometry showed that the cell proliferation index (PI ) of the subgroups except the 5×10-5 mol·L-1 BPA subgroup presented a rising tendency compared with the control group, but there was no significant difference. The proportion of cells in S phase of the 5×10-6 mol·L-1 BPA subgroup was significantly higher than that of the control group (P < 0.05). Compared with the control group, the expression level of Cyclin D1 protein decreased and the expression level of P21 protein increased in the 5×10-5 mol·L-1 BPA subgroup; in contrast, the expression level of Cyclin D1 protein increased and the expression level of P21 protein decreased in the 5×10-8 and 5×10-9 mol·L-1 BPA subgroups (P < 0.05).Conclusion BPA has dual effects on the proliferation of KTC-1 cells. High-dose BPA has a toxic effect on KTC-1 cells and leads to cell death; low-dose BPA promotes the proliferation of KTC-1 cells. Different concentrations of BPA can either promote or inhibit the proliferation of KTC-1 cells by regulating the expression of Cyclin D1 and P21.
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