碘过量对FRTL细胞过氧化损伤的实验研究

发布时间：2018-03-02 05:16

本文关键词： 碘过量 FRTL细胞线粒体超氧化物凋亡细胞色素C　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究过量碘化钾(potassium iodide, KI)对Fisher大鼠甲状腺细胞(Fisher rat thyroid cell line, FRTL)线粒体超氧化物生成、细胞活力及细胞凋亡的影响,探讨碘过量对FRTL细胞过氧化损伤的发生、发展过程和机制,并用促甲状腺激素(thyrotropin, TSH)或丙基硫氧嘧啶(propylthiouracil, PTU)进行干预,研究其对碘过量导致的FRTL细胞过氧化损伤的影响。 1.应用甲基噻唑基四唑(methyl thiazolyl tetrazolium, MTT)比色法检测不同浓度(10-7mol/L～10-2mol/L) KI对FRTL细胞活力的影响,相应浓度氯化钾(potassium chloride, KCl)作为渗透压对照。 2.分别用10-4 mol/L、10-2mol/L KI处理2h、4h、24h,利用MitoSOX通过流式细胞术和荧光显微镜检测FRTL细胞线粒体超氧化物生成,利用免疫细胞化学法和酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)检测FRTL细胞内细胞色素C(cytochrome c, cyt c)释放,利用乳酸脱氢酶(lactate dehydrogenase, LDH)测定试剂盒检测细胞培养液上清的LDH活力,利用碘化丙啶(propidium iodide, PI)通过流式细胞术检测死亡细胞百分比(percent of dead cells),DNA琼脂糖电泳检测DNA断裂片段,透射电镜观察超微结构改变。 3.用10"4mol/LKI加或不加10U/L TSH处理24h,检测FRTL细胞线粒体超氧化物生成和细胞活力。 4.用10-4mol/LKI加或不加300μmol/L PTU处理2h、24h,检测FRTL细胞线粒体超氧化物生成和细胞活力。 1.处理30min,10-7～10-2mol/LKI或KCl不影响FRTL细胞活力(p0.05)；处理24h,10-5～10-2mol/LKI明显降低FRTL细胞活力(p0.05),10-7～10-2 mol/LKCl不影响细胞活力(p0.05)。 2.碘过量对FRTL细胞过氧化损伤的时效性 (1)处理2h、4h、24h,FRTL细胞内超氧化物生成增加,以2h增加最明显。 (2)处理2h、4h、24h,cyt c释放增加(p0.01),4h最明显。 (3)处理4h、24h,培养液上清LDH活力明显增加(p0.01),死亡细胞百分比升高。 (4)处理24h,出现DNA ladder。 (5)电镜下处理2h,线粒体肿胀,4h细胞膜、线粒体膜溶解、内质网扩张、髓磷体形成,24h凋亡小体出现。 3. TSH或PTU对碘过量导致的FRTL细胞过氧化损伤的影响 (1)处理24h,KI+TSH组线粒体超氧化物生成明显低于KI组(p0.05),细胞活力明显高于KI组(p0.05)。 (2)处理2h,KI+PTU组线粒体超氧化物生成明显低于KI组(p0.01),处理24h,KI+PTU组细胞活力明显高于KI组(p0.05)。 1.KI对FRTL细胞活力的影响具有浓度依赖性。 2.碘过量对FRTL细胞过氧化损伤具有时效性,2h导致线粒体氧化损伤,伴有cyt c释放,4h细胞膜受损,线粒体膜溶解,24h细胞损伤进一步加重,并出现细胞凋亡。 3. TSH(10U/L)在一定程度上可以减轻碘过量对FRTL细胞的过氧化损伤。 4.PTU可以减轻碘过量对FRTL细胞过氧化损伤。
[Abstract]:To study the effects of excessive potassium iodium iodide (Ki) on the production of mitochondrial superoxide, cell viability and apoptosis in Fisher rat thyroid cells, and to explore the occurrence, development and mechanism of excessive iodine on the peroxidation injury of FRTL cells. The effects of thyrotropin (TSH) or propylthiouracil (PTU) on the peroxidation of FRTL cells induced by iodine excess were studied. 1.Methylthiazolyl thiazolyl tetrazolium (MTT) colorimetric assay was used to detect the effects of different concentrations of 10 ~ (-7) mol / L ~ (-2) mol / L ~ (-1) Ki on the viability of FRTL cells, and potassium chloride potassium chloride (KCl) was used as osmotic pressure control. 2.It was treated with 10-4 mol / L 10-2mol / L Ki for 2 h ~ 4 h ~ (-1) for 24 h respectively. Mitochondrial superoxide production in FRTL cells was detected by flow cytometry and fluorescence microscope by MitoSOX. The release of cytochrome C (cyt c) from FRTL cells was detected by immunocytochemistry and enzyme linked immunosorbent assay.Lactic dehydrogenase lactate dehydrogenase (LDH) was used to detect the activity of LDH in supernatant of cell culture medium. Using propidium iodide (Pi), the percent percent of dead cells were detected by flow cytometry to detect the fragmentation of DNA fragments by agarose electrophoresis. The ultrastructural changes were observed by transmission electron microscopy (TEM). 3. FRTL cells were treated with 10 "4mol / L Ki plus or without 10 U / L TSH for 24 h to detect mitochondrial superoxide production and cell viability. 4. FRTL cells were treated with 10-4 mol / L LKI for 2 h or without 300 渭 mol/L PTU for 24 h to detect mitochondrial superoxide production and cell viability. 1. Treatment of 10-7 mol / L LKI or KCl for 30 min did not affect the viability of FRTL cells (p0.05), but the treatment of 10-5 mol / LKI for 24 h significantly decreased the viability of FRTL cells (p0.05 ~ 10-7 / 10 ~ (-2) mol/LKCl), and did not affect the viability of FRTL cells (P _ (0.05) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1)). 2. Effect of iodine excess on peroxidation injury of FRTL cells. 1) the superoxide production in FRTL cells was increased at 2 h, 2 h and 24 h, especially at 2 h. (2) the release of cyt c increased at 4 h after treatment for 4 h and increased significantly for 4 h after treatment. (3) the activity of LDH in supernatant of culture medium increased significantly at 4 h or 24 h, and the percentage of dead cells increased. 4) DNA ladder appeared after 24 h treatment. Under electron microscope for 2 h, mitochondria swelled at 4 h of cell membrane, mitochondria membrane dissolved, endoplasmic reticulum dilated, myelophosphorus formed 24 h apoptotic corpuscles appeared. 3. Effects of TSH or PTU on peroxidation injury of FRTL cells induced by iodine excess. (1) the mitochondrial superoxide production in Ki TSH group was significantly lower than that in Ki group at 24 h, and the cell viability was significantly higher than that in Ki group. (2) the mitochondrial superoxide production in Ki PTU group was significantly lower than that in Ki group at 2 h, and the cell viability in Ki PTU group was significantly higher than that in Ki group at 24 h. 1. The effect of Ki on FRTL cell viability was concentration-dependent. 2. The oxidative damage of FRTL cells was induced by iodine overdose for 2 h, accompanied by the damage of cyt c release cell membrane for 4 h, the damage of mitochondria membrane dissolved for 24 h was further aggravated, and apoptosis occurred. 3. TSH 10 U / L can alleviate the peroxidation damage of FRTL cells induced by iodine excess to some extent. 4. PTU could reduce the peroxidation injury of FRTL cells induced by iodine excess.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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