碘过量对大鼠甲状腺细胞线粒体的早期影响

Peroxide effects of iodide excess on mitochondria in Fischer rat thyroid cell line in the early period

目的探讨碘过量对Fischer大鼠甲状腺细胞（FRTL）线粒体过氧化损伤的早期影响。方法分别用含0．O（对照）、0．1mmol／L碘化钾（rd）的培养基培养FRTL细胞2、4、24h。利用线粒体超氧化物指示剂（MitoSOX）通过流式细胞术和荧光显微镜检测线粒体超氧化物生成，免疫细胞化学法检测细胞色素C（cytC）释放，比色法检测细胞培养液上清乳酸脱氢酶（LDH）活力，利用碘化丙啶通过流式细胞术检测死亡细胞百分比，DNA电泳检测凋亡DNA片段。结果0．1mmol／LKI处理2、4、24h时，FRTL细胞线粒体超氧化物生成均较对照组升高，以2h升高最明显；cytC强阳性细胞率分别为（35．3±3．6）％、（45．8±5．5）％、（30．3±6．1）％，明显高于对照组[（14．8±1．2）％，P均〈0．05]；LDH活力分别为（1．85±0．32）、（6．63±0．42）、（8．35±0．34）U／mg，其中4、24h时明显高于对照组[（0．89±0．04）U／mg，P均〈0．05]；对照组和0．1mmol／LKI处理2、4、24h组死亡细胞百分比分别为4．66％、7．52％、9．29％、13．71％；0．1mmol／LKI处理24h时，出现DNAladder。结论碘过量处理2h可引起FRTL细胞线粒体过氧化损伤。24h可引起细胞凋亡。

Objective To investigate the peroxide effects of iodide excess on mitochondria in Fischer rat thyroid cell line （FRTL） in the early period. Methods After treatment with 0.0 mmol/L（control group） or 0.1 mmol/L potassium iodide（KI） for 2, 4 and 24 h, respectively, changes of mitochondrial superoxide formation were assayed by flow cytometry and fluorescence microscopy using mitochondria-targeted hydroethidine（MitoSOX）. The cytochrome c （cyt c） released from mitochondria to cytoplasm was detected by immunocytochemistry. The lactate dehydrogenase （LDH） released into supernatant was measured by a LDH kit using colorimetry. The percent of dead ceils was assayed by flow cytometry using propidium iodide （PI）. DNA with loading buffer was separated in 1% agarose gel. Results Mitochondrial superoxide production in FRTL cells treated with 0.1 mmol/L KI was increased at 2, 4 and 24 h, especially at 2 h. The rates of cyc c protein immunity positive cells at 2, 4 and 24 h in 0.1 mmol/L KI group were （35.3± 3.6）%, （45.8 ±5.5）% and （30.3±6.1）%, respectively, which were significantly higher than that in control group[ （14.8± 1.2）%, all P 〈 0.05]. The LDHs released into supernatant at 2, 4 and 24 h in 0.1 mmol/L KI group were （1.85 ± 0.32）, （6.63 ± 0.42） and （8.35± 0.34）U/mg, and these values at 4 and 24 h in 0.1 mmol/L KI group were significantly higher than that of control group[ （0.89 ±0.04）U/mg, all P 〈 0.05 ]. After incubation with 0.1 mmol/L KI for 2, 4 and 24 h, the percentages of dead FRTL ceils were 7.52%, 9.29% and 13.71%, respectively, while that of control group was 4.66%. After FRTL cells were treated with 0.1 mmol/L KI for 24 h, DNA ladder appeared. Conclusion Iodide excess （0.1 mmol/L） can cause mitochondrial peroxide injury in FRTL cells at 2 h and ceil apoptosis at 24 h.