过氯酸铵对甲状腺细胞毒作用机制的研究

Study on mechanism of thyroid cytotoxicity of ammonium perchlorate

目的探讨过氯酸铵（AP）对甲状腺细胞的毒作用机制。方法将甲状腺体外细胞培养到一定阶段，分别给予AP浓度为0、5、10、20、40、60mmol／L的培养液进行细胞染毒，收集培养的细胞和上清液做以下指标测定。用噻唑蓝比色法（MTT法）测定细胞增殖，用流式细胞技术检测法测定细胞凋亡，用酶联免疫吸附试验（ELISA）测定甲状腺球蛋白（取）浓度，用比色法测定乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、丙二醛（MDA）等指标。结果60mmol／LAP染毒组细胞12、24、48、72h时，存活率分别为74．93％、42．26％、2．66％和0．99％，40mmol／LAP染毒组细胞24、48、72h时，细胞存活率分别为73．15％、30．91％和3．03％，与对照组（100％）比较，差异均有统计学意义（P＜0．05或P〈0．01）。各染毒组细胞总凋亡率均较对照组明显增加，20、40、60mmol/L剂量组细胞早期凋亡率分别为15．70％、15．84％和16．96％，较对照组（9．54％）明显增加（P〈0．05或P〈0．01）；60mmol／L剂量组细胞晚期凋亡率为16．54％，较对照组（6．11％）明显增加，差异均有统计学意义（P〈0．05或P〈0．01）。40mmol／L染毒组细胞LDH活力为0．70U／ml，较对照组（0．55U／m1）明显升高，差异有统计学意义（P〈0．01）；5mmol／L染毒组细胞MDA含量为1．08mmol，L，较对照组（2．36mmol／L）降低，差异亦有统计学意义（P〈0．05）。结论AP作用于甲状腺细胞，可明显改变细胞形态，降低细胞存活率，这可能与AP抑制甲状腺细胞增殖，诱导甲状腺细胞凋亡，破坏甲状腺细胞膜完整性有关。AP对甲状腺细胞氧化损伤并不明显。

Objective To investigate the mechanism of thyroid cytotoxicity mechanism of ammonium perchlorate （AP）. Methods Thyroid cells were cultured in vitro to a certain stage and then exposed to AP （0, 5, 10, 20, 40, and 60 mmol/L） in culture solution; the cultured cells and supernatant were collected. Cell viability was measured by MTF assay; cell apoptosis was determined by flow cytometry; the concentration of thyroglobulin was measured by enzyme-linked immunosorbent assay; the lactate dehydrogenase （LDH） activity, superoxide dismutase （SOD） activity, malondialdehyde （MDA） level, and so on were measured by colorimetry. Results The cells exposed to 60 mmol/L AP for 12, 24, 48, and 72 h had cell viabilities of 74.93%, 42.26%, 2.66%, and 0.99%, respectively, and the ceils exposed to 40 mmol/L AP for 24, 48, and 72 h had cell viabilities of 73.15%, 30.91%, and 3.03%, respectively, all significantly lower than that of the control group （100%） （P〈 0.05 or P〈0.01）. The overall apoptosis rate of all AP-exposed cells was significantly higher than that of the control group; the cells exposed to 20, 40, and 60 mmol/L AP had early apoptosis rates of 15.70%, 15.84%, and 16.96%, respectively, significantly higher than that of the control group （9.54%） （P〈0.05 or P〈0.01 ）; the cells exposed to 60 mmol/L AP had a late apoptosis rate of 16.54%, significantly higher than that of the control group （6.11%） （P〈0.05 or P〈0.01 ）. The cells exposed to 40 mmol/L AP had a significantly higher LDH activity than the control group （0.70 U/ml vs 0.55 U/ml, P〈0.01）. The cells exposed to 5 mmol/L AP had a significantly higher MDA level than the control group （1.08 mmol/L vs 2.36 mmol/L, P〈0.05）. Conclusion AP can markedly change the cell morphology and decrease the cell viability of thyroid cells, which may be because AP inhibits cell proliferation, induces cell apoptosis, and destroys cell membranes. However, AP does not result in significant oxidative damage to thyroid cells.