高碘诱导Nthy-ori 3-1细胞凋亡机制的研究

发布时间：2018-07-03 04:09

本文选题：高碘 + Nthy-ori3-1细胞　；参考：《天津医科大学》2012年硕士论文

【摘要】：高碘摄入可以引起甲状腺结构变化和形态异常,甚至造成甲状腺功能减退及功能亢进等疾病。高碘摄入对甲状腺功能损伤的机制至今仍不明了,存在很多争议。近年来,不同剂量的碘诱导甲状腺细胞凋亡成为国内外学者的研究热点：Vitale M等通过体外实验证实,高碘可以通过影响活性氧的产生,抑制甲状腺细胞生长,甚至造成细胞凋亡和坏死。此外,高碘还可干扰甲状腺细胞周期,使细胞停滞在G0/G1期和G2/M期,抑制细胞增殖；徐健和林来祥等研究高碘的毒性效应,发现过量碘可通过调节Fas、FasL、Bcl2及Bax基因的表达促进甲状腺细胞凋亡的发生。内质网位于细胞核附近的胞质区域,是哺乳动物细胞最重要的Ca2+贮存器,也是蛋白质合成与翻译后修饰的重要场所。缺氧、氧化应激、化学毒物等均可引起内质网的微环境改变,出现错误折叠和未折叠蛋白在腔内聚集,伴随Ca2+平衡紊乱,导致内质网应激(Endoplasmic Reticulum Stress, ERS)。研究表明,导致细胞凋亡的三条经典通路：线粒体通路、死亡受体通路和内质网通路存在着千丝万缕的联系,三者在某种程度上会相互影响。研究者们认为高碘诱导甲状腺细胞凋亡的发生主要通过线粒体途径和死亡受体途径,而且各类实验也已取得了各种研究结果,但是高碘能否通过内质网途径诱导甲状腺细胞凋亡,目前未见报道。目的研究高浓度碘化钾(Potassium Iodide, KI)对人正常甲状腺细胞系Nthy-ori3-1(Human Thyroid Follicular Epithelial, Nthy-ori3-1)细胞凋亡、活性氧(Reactive Oxygen Species, ROS)产生和内质网相关基因及蛋白表达的影响,探讨高碘对Nthy-ori3-1细胞凋亡、内质网应激的发生、发展过程及机制。方法 1.分别采用1、10、50mmol/L KI染毒Nthy-ori3-1细胞,以蒸馏水为溶剂对照,应用乳酸脱氢酶(Lactate Dehydrogenase, LDH)试剂盒检测不同浓度的KI对Nthy-ori3-1细胞LDH漏出率的影响； 2.利用碘化丙啶(Propidium Iodide, PI)通过流式细胞术检测不同浓度KI对Nthy-ori3-1细胞周期的影响和凋亡细胞百分比(Apoptosis Rate)。运用2’-7’-二氯荧光黄双乙酸盐(2'-7'Dichlorodihydrofluorescein Diacetate, DCFH-DA)荧光探针法通过流式细胞术检测不同浓度KI对Nthy-ori3-1细胞活性氧的产生情况； 3.体外培养的Nthy-ori3-1细胞暴露于不同浓度的KI (1、10、50mmol/L),以蒸馏水为溶剂对照组,染毒24h后,用RT-PCR检测内质网应激(ERS)相关基因GRP78、IRE1、XBP-1、CHOP mRNA表达情况； 4.体外培养的Nthy-ori3-1细胞暴露于不同浓度的KI (1、10、50mmol/L),以蒸馏水为溶剂对照组,染毒24h后,用Western blot检测内质网应激(ERS)相关蛋白GRP78、IRE1、CHOP的表达情况。结果 1.与对照组、1、10mmol/L KI染毒组相比,50mmol/L KI染毒组乳酸脱氢酶(LDH)漏出率和细胞凋亡率明显升高,差异均具有统计学意义(P0.05); 2.与对照组相比,1、10、50mmol/L KI染毒组细胞ROS差异均无统计学意义(P0.05)； 3.与对照组、1、10mmol/L KI染毒组相比,50mmol/L KI染毒组Go/G1期细胞数明显上升,S期细胞数明显减少,差异具有统计学意义(P0.05)； 4.染毒24h后,采用RT-PCR检测内质网应激(ERS)相关基因GRP78、IRE1、XBP-1(s)、CHOP mRNA。与对照组相比,1、10、50mmol/L KI染毒组GRP78、IRE1mRNA表达升高,但差异无统计学意义(P0.05)；对照组与各染毒组XBP-1(s)表达均未检测到；各染毒组CHOP mRNA表达显著升高(P0.05),但各组之间的差异无统计学意义(P0.05)； 5.染毒24h后,采用Western blot检测内质网应激(ERS)GRP78、IRE1、 CHOP相关蛋白,结果显示：对照组、1、10、50mmol/L KI染毒组蛋白表达差异均无统计学意义(P0.05)。结论 1.用来评价细胞膜完整性指标的LDH漏出率,在高碘的情况下显著增加,且在一定程度上存在随染毒剂量上升而升高的趋势。高碘可干扰甲状腺细胞周期,使细胞停滞在Go/G1期,诱导体外培养的Nthy-ori3-1细胞凋亡率升高； 2.高碘可能不会导致Nthy-ori3-1细胞ROS水平升高,诱导氧化应激反应发生,可能与细胞种属有关,具体的机制还需要进一步的深入研究和探讨； 3.高碘不引起Nthy-ori3-1细胞内质网应激反应,后者可能不参与高碘诱导的Nthy-ori3-1细胞凋亡过程。项目来源：国家自然科学基金(30972555)
[Abstract]:High iodine intake may cause abnormal thyroid structure and abnormal morphology , even cause hypothyroidism and hyperfunction . In recent years , the mechanism of high iodine intake on thyroid function injury is still unknown , there are many disputes . In recent years , various doses of iodine - induced apoptosis of thyroid cells have become a hot spot at home and abroad . In recent years , high iodine can inhibit the growth of thyroid cells and even cause apoptosis and necrosis .
Xu Jian and Lin Xiangxiang studied the toxic effects of high iodine . It was found that excess iodine could promote the apoptosis of thyroid cells by regulating the expression of Fas , FasL , Bcl 2 and Bax genes .

The endoplasmic reticulum ( ER ) , which is the most important Ca2 + reservoir in mammalian cells , is the most important Ca2 + reservoir in mammalian cells , and is also an important place for protein synthesis and post - translational modification . Hypoxia , oxidative stress , chemical poison and the like can cause the microenvironment of the endoplasmic reticulum to change , and the presence of error folding and unfolded protein accumulation in the cavity , accompanied by a disturbance of Ca2 + balance , leads to endoplasmic reticulum stress ( ERS ) . The study shows that there are three classical pathways leading to apoptosis : mitochondrial pathway , death receptor pathway and endoplasmic reticulum ( ER ) pathway exist in the presence of thousands of strands , which can interact to some extent . The researchers believe that high iodine induces apoptosis of thyroid cells mainly through the pathway of mitochondrial pathway and death receptor , and the results of various experiments have been obtained , but whether high iodine can induce apoptosis through the endoplasmic reticulum pathway is not reported .

Purpose

The effects of potassium iodide ( KI ) on the apoptosis of human normal thyroid cell line Nthy - ori3 - 1 ( Nthy - ori3 - 1 ) , the production of reactive oxygen species ( ROS ) and the expression of related genes and proteins in the endoplasmic reticulum were investigated .

method

1 . The effects of KI on LDH leakage rate in Nthy - ori3 - 1 cells were detected with lactate dehydrogenase ( LDH ) kit by using Nthy - ori3 - 1 cells of 1 , 10 , 50 mmol / L KI and distilled water as solvent control .

2 . The effect of KI on the cell cycle of Nthy - ori3 - 1 and the percentage of apoptotic cells ( apoptosis rate ) of KI on Nthy - ori3 - 1 cell cycle were detected by flow cytometry using propidium iodide ( PI ) . The production of active oxygen in Nthy - ori3 - 1 cells was detected by flow cytometry by flow cytometry using 2 ' -7 ' - dichlorofluorescein diacetate ( DCFH - DA ) fluorescence probe method .

3 . The Nthy - ori3 - 1 cells cultured in vitro were exposed to KI ( 1 , 10 , 50 mmol / L ) at different concentrations , and the expression of GRP78 , IRE1 , XBP - 1 and CHOP mRNA was detected by RT - PCR in the control group of distilled water .

4 . The Nthy - ori3 - 1 cells cultured in vitro were exposed to KI ( 1 , 10 , 50 mmol / L ) at different concentrations , and the expression of GRP78 , IRE1 , CHOP in the endoplasmic reticulum stress ( ERS ) related protein was detected by Western blot after 24 hours of exposure to distilled water as the solvent control group .

Results

1 . Compared with the control group , the leakage rate of lactate dehydrogenase ( LDH ) and apoptosis rate in 50 mmol / L KI group were significantly higher than those in control group , 1 , 10 mmol / L KI group ( P0.05 ) .

2 . Compared with the control group , there was no significant difference in the content of ROS in the 1 , 10 , 50 mmol / L KI staining group ( P0.05 ) .

3 . Compared with the control group , the number of G0 / G1 phase cells increased and the number of S phase cells decreased significantly compared with the control group , 1 , 10 mmol / L KI staining group ( P0.05 ) .

4 . The expression of GRP78 , IRE1 , XBP - 1 ( s ) and CHOP mRNA was detected by RT - PCR after 24 h of exposure . Compared with the control group , the expression of GRP78 and IRE1 mRNA in 1 , 10 , 50 mmol / L KI were increased , but there was no statistical significance ( P0.05 ) .
The expression of XBP - 1 ( s ) in the control group was not detected .
CHOP mRNA expression in each group increased significantly ( P0.05 ) , but there was no significant difference between the groups ( P0.05 ) .

5 . After incubation for 24 h , the expression of GRP78 , IRE1 , CHOP protein was detected by Western blot . The results showed that there was no significant difference in the expression of protein in the control group , 1 , 10 , 50 mmol / L KI ( P0.05 ) .

Conclusion

1 . LDH leakage rate , which was used to evaluate the cell membrane integrity index , was significantly increased in the case of high iodine , and there was a tendency to increase with the increase of the dosage of the dye . High iodine could interfere with the cell cycle of the thyroid gland and arrest the apoptosis rate of Nthy - ori3 - 1 cells cultured in vitro .

2 . High iodine may not result in the increase of ROS level in Nthy - ori3 - 1 cell , induce oxidative stress reaction , which may be related to cell species , and the specific mechanism needs further research and discussion ;

3 . High iodine does not induce the endoplasmic reticulum stress response of Nthy - ori3 - 1 cells , which may not be involved in the process of apoptosis of Nthy - ori3 - 1 cells induced by high iodine . Source : National Natural Science Foundation ( 30972555 )
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151.41

【参考文献】