本文選題：高碘 + Nthy-ori3-1細(xì)胞�。� 參考：《天津醫(yī)科大學(xué)》2012年碩士論文

【摘要】：高碘攝入可以引起甲狀腺結(jié)構(gòu)變化和形態(tài)異常,甚至造成甲狀腺功能減退及功能亢進等疾病。高碘攝入對甲狀腺功能損傷的機制至今仍不明了,存在很多爭議。近年來,不同劑量的碘誘導(dǎo)甲狀腺細(xì)胞凋亡成為國內(nèi)外學(xué)者的研究熱點：Vitale M等通過體外實驗證實,高碘可以通過影響活性氧的產(chǎn)生,抑制甲狀腺細(xì)胞生長,甚至造成細(xì)胞凋亡和壞死。此外,高碘還可干擾甲狀腺細(xì)胞周期,使細(xì)胞停滯在G0/G1期和G2/M期,抑制細(xì)胞增殖；徐健和林來祥等研究高碘的毒性效應(yīng),發(fā)現(xiàn)過量碘可通過調(diào)節(jié)Fas、FasL、Bcl2及Bax基因的表達促進甲狀腺細(xì)胞凋亡的發(fā)生。 內(nèi)質(zhì)網(wǎng)位于細(xì)胞核附近的胞質(zhì)區(qū)域,是哺乳動物細(xì)胞最重要的Ca2+貯存器,也是蛋白質(zhì)合成與翻譯后修飾的重要場所。缺氧、氧化應(yīng)激、化學(xué)毒物等均可引起內(nèi)質(zhì)網(wǎng)的微環(huán)境改變,出現(xiàn)錯誤折疊和未折疊蛋白在腔內(nèi)聚集,伴隨Ca2+平衡紊亂,導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic Reticulum Stress, ERS)。研究表明,導(dǎo)致細(xì)胞凋亡的三條經(jīng)典通路：線粒體通路、死亡受體通路和內(nèi)質(zhì)網(wǎng)通路存在著千絲萬縷的聯(lián)系,三者在某種程度上會相互影響。研究者們認(rèn)為高碘誘導(dǎo)甲狀腺細(xì)胞凋亡的發(fā)生主要通過線粒體途徑和死亡受體途徑,而且各類實驗也已取得了各種研究結(jié)果,但是高碘能否通過內(nèi)質(zhì)網(wǎng)途徑誘導(dǎo)甲狀腺細(xì)胞凋亡,目前未見報道。 目的 研究高濃度碘化鉀(Potassium Iodide, KI)對人正常甲狀腺細(xì)胞系Nthy-ori3-1(Human Thyroid Follicular Epithelial, Nthy-ori3-1)細(xì)胞凋亡、活性氧(Reactive Oxygen Species, ROS)產(chǎn)生和內(nèi)質(zhì)網(wǎng)相關(guān)基因及蛋白表達的影響,探討高碘對Nthy-ori3-1細(xì)胞凋亡、內(nèi)質(zhì)網(wǎng)應(yīng)激的發(fā)生、發(fā)展過程及機制。 方法 1.分別采用1、10、50mmol/L KI染毒Nthy-ori3-1細(xì)胞,以蒸餾水為溶劑對照,應(yīng)用乳酸脫氫酶(Lactate Dehydrogenase, LDH)試劑盒檢測不同濃度的KI對Nthy-ori3-1細(xì)胞LDH漏出率的影響； 2.利用碘化丙啶(Propidium Iodide, PI)通過流式細(xì)胞術(shù)檢測不同濃度KI對Nthy-ori3-1細(xì)胞周期的影響和凋亡細(xì)胞百分比(Apoptosis Rate)。運用2’-7’-二氯熒光黃雙乙酸鹽(2'-7'Dichlorodihydrofluorescein Diacetate, DCFH-DA)熒光探針法通過流式細(xì)胞術(shù)檢測不同濃度KI對Nthy-ori3-1細(xì)胞活性氧的產(chǎn)生情況； 3.體外培養(yǎng)的Nthy-ori3-1細(xì)胞暴露于不同濃度的KI (1、10、50mmol/L),以蒸餾水為溶劑對照組,染毒24h后,用RT-PCR檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)相關(guān)基因GRP78、IRE1、XBP-1、CHOP mRNA表達情況； 4.體外培養(yǎng)的Nthy-ori3-1細(xì)胞暴露于不同濃度的KI (1、10、50mmol/L),以蒸餾水為溶劑對照組,染毒24h后,用Western blot檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)相關(guān)蛋白GRP78、IRE1、CHOP的表達情況。 結(jié)果 1.與對照組、1、10mmol/L KI染毒組相比,50mmol/L KI染毒組乳酸脫氫酶(LDH)漏出率和細(xì)胞凋亡率明顯升高,差異均具有統(tǒng)計學(xué)意義(P0.05); 2.與對照組相比,1、10、50mmol/L KI染毒組細(xì)胞ROS差異均無統(tǒng)計學(xué)意義(P0.05)； 3.與對照組、1、10mmol/L KI染毒組相比,50mmol/L KI染毒組Go/G1期細(xì)胞數(shù)明顯上升,S期細(xì)胞數(shù)明顯減少,差異具有統(tǒng)計學(xué)意義(P0.05)； 4.染毒24h后,采用RT-PCR檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)相關(guān)基因GRP78、IRE1、XBP-1(s)、CHOP mRNA。與對照組相比,1、10、50mmol/L KI染毒組GRP78、IRE1mRNA表達升高,但差異無統(tǒng)計學(xué)意義(P0.05)；對照組與各染毒組XBP-1(s)表達均未檢測到；各染毒組CHOP mRNA表達顯著升高(P0.05),但各組之間的差異無統(tǒng)計學(xué)意義(P0.05)； 5.染毒24h后,采用Western blot檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)GRP78、IRE1、 CHOP相關(guān)蛋白,結(jié)果顯示：對照組、1、10、50mmol/L KI染毒組蛋白表達差異均無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 1.用來評價細(xì)胞膜完整性指標(biāo)的LDH漏出率,在高碘的情況下顯著增加,且在一定程度上存在隨染毒劑量上升而升高的趨勢。高碘可干擾甲狀腺細(xì)胞周期,使細(xì)胞停滯在Go/G1期,誘導(dǎo)體外培養(yǎng)的Nthy-ori3-1細(xì)胞凋亡率升高； 2.高碘可能不會導(dǎo)致Nthy-ori3-1細(xì)胞ROS水平升高,誘導(dǎo)氧化應(yīng)激反應(yīng)發(fā)生,可能與細(xì)胞種屬有關(guān),具體的機制還需要進一步的深入研究和探討； 3.高碘不引起Nthy-ori3-1細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),后者可能不參與高碘誘導(dǎo)的Nthy-ori3-1細(xì)胞凋亡過程。項目來源：國家自然科學(xué)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 )
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R151.41

【參考文獻】

相關(guān)期刊論文 前7條

1 付建芳,姬秋和,黃威權(quán),張南雁;橋本甲狀腺炎中TRAIL及其死亡受體DR4,DR5的表達[J];第四軍醫(yī)大學(xué)學(xué)報;2002年10期

2 鄭合明,王羽,王明臣;不同碘濃度對小鼠抗氧化能力的研究[J];中國地方病防治雜志;2002年04期

3 肖建英;田英娟;朱燕凌;許珂玉;王翠瑤;;過量碘對原代培養(yǎng)的豬甲狀腺細(xì)胞凋亡的誘導(dǎo)作用[J];中國地方病防治雜志;2006年02期

4 謝靜;羅凌惠;薛秋紅;龔樹生;;內(nèi)質(zhì)網(wǎng)應(yīng)激在老年大鼠耳蝸細(xì)胞凋亡中的作用[J];中國耳鼻咽喉頭頸外科;2008年10期

5 黃穎泓;楊立勇;嚴(yán)孫杰;張聲;;碘化鈉和IL-1β對人甲狀腺上皮細(xì)胞TRAIL表達的影響[J];福建醫(yī)科大學(xué)學(xué)報;2011年05期

6 秦俊法;樓蔓藤;李增禧;肖益新;;高碘甲狀腺病:一個不容忽視的公共衛(wèi)生問題[J];廣東微量元素科學(xué);2010年05期

7 林來祥;孫毅娜;張璐;閻玉芹;陳祖培;;短期碘缺乏和碘過量對大鼠甲狀腺細(xì)胞凋亡和增殖的影響[J];環(huán)境與健康雜志;2007年08期

，

本文編號：2092335