KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 碘代消毒副产物细胞毒性作用及其相关机制研究 | |
| 陈娇 | |
| Subtype | 硕士 |
| Thesis Advisor | 梁岩 |
| 2018-06-01 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 新疆乌鲁木齐 |
| Degree Discipline | 理学硕士 |
| Keyword | 碘代消毒副产物 细胞毒性 氧化损伤 细胞凋亡 iodinated disinfection by-products cytotoxicity oxidative damage apoptosis |
| Abstract | 碘代消毒副产物（Iodo-DBPs）是一种新型的未受控消毒副产物。目前已鉴定出 20 种碘代消毒副产物，其中 15 种已在氯化消毒水中检测到，大量的毒理学研究表明，碘代消毒副产物（Iodo-DBPs）与氯代和溴代消毒副产物相比，具有更强的细胞毒性和遗传毒性。近年来碘代消毒副产物的形成及毒理效应研究受到越来越多学者的关注。本文选用 HepG -2 细胞和 CaCo-2 细胞对其中 8 种Iodo-DBPs 毒性作用及其相关机制进行了研究。 结果如下：1） 通过倒置显微镜观察碘乙腈（IAN）对两种细胞形态的变化，结果表明6μM IAN 染毒 HepG-2 和 CaCo-2 细胞 24h 后，细胞形态均发生明显变化，处理组 HepG-2 细胞皱缩变圆明显，且细胞间连接松弛；处理组 CaCo-2 细胞正常形态消失，细胞边缘缩小模糊，可见大量细胞碎片和悬浮的死细胞，细胞受到严重的损伤。通过 MTT 实验表明， 8 种 Iodo-DBPs 都会对 HepG-2 细胞和 CaCo-2 细胞的细胞增值率产生影响，与浓度和时间的变化有着明显的效应关系。暴露浓度增加或暴露时间延长，都会使细胞增殖率呈现明显下降趋势。2） 通过检测细胞内活性氧（ROS）、丙二醛（MDA）和总谷胱甘肽（GSH）产生量， RT-PCR 检测 NRF-2 及其下游抗氧化基因 GCLC 的表达量， Western Blot检测 NRF-2 和 GCLC 蛋白表达水平，了解 8 种 Iodo-DBPs 对细胞氧化损伤影响。结果表明 8 种 Iodo-DBPs 均会诱导 HepG-2 细胞和 CaCo-2 细胞产生大量活性氧ROS，并且生成量的高低与细胞毒性大小趋势一致； 8 种 Iodo-DBPs 暴露 HepG-2细胞后，丙二醛含量明显升高，且 8 种 Iodo-DBPs 暴露于 HepG-2 细胞产生 MDA大小顺序为 IAA > IAN > BCIM> TIM> BDIM> DBIM> CDIM> DCIM；随着Iodo-DBPs 浓度的增加，细胞内 GSH 先是处于缓慢的减少状态，当 Iodo-DBPs浓度升高到一定值时，细胞内 GSH 含量也急剧下降； Iodo-DBPs 会致使 HepG -2细胞启动控制氧化还原反应的元件 Nrf-2，并调控下游基因 GCLC 的表达以维持细胞内氧化-还原相对稳定的状态并且促使胞内氧化损伤的发生。3） 通过流式细胞仪检测细胞凋亡率， RT-PCR 检测促凋亡基因 Bax 和抑凋亡基因 Bcl-2 表达水平，以及 Western Blot 检测 Bax、 Bcl-2、 caspase-3 蛋白表达水平，研究结果表明，暴露于 Iodo-DBPs 中的 HepG-2 细胞和 CaCo-2 细胞会引起细胞凋亡，细胞凋亡受暴露剂量影响，浓度越高，凋亡现象越明显。其中，HepG-2 细胞凋亡率的大小排序为： IAN > IAA > DCIM > CDIM > BCIM >DBIM > TIM >BDIM； CaCo-2 细胞凋亡率的大小排序为： IAA > IAN > BDIM >DCIM > DBIM > CDIM > TIM >BCIM。高剂量的 Iodo-DBPs 是加剧 HepG-2 细胞和 CaCo-2 细胞凋亡的主要原因，且 CaCo-2 细胞的凋亡率明显高于 HepG-2 细胞凋亡率。 8 种 Iodo-DBPs 急性染毒 24 小时对 HepG-2 细胞中基因 Bax 和 Bcl-2的表达均上调，且 Bax/Bcl-2 值相比对照组均增加。总结这 8 种 Iodo-DBPs 诱导的凋亡是通过线粒体通路形成的。 Iodo-DBPs 暴露可导致 Bax 和 Caspase-3 蛋白表达上调，且呈现浓度依赖性增加。 Iodo-DBPs 暴露可导致抑凋亡基因 Bcl-2 蛋白表达下调。可以推测这 8 种 Iodo-DBPs 使两种细胞产生氧化应激有可能是诱导细胞凋亡的原因。 |
| Other Abstract | Iodo-DBPs is a new uncontrolled disinfection byproduct.At present, 20 iodinateddisinfection by-products have been identified, of which 15 have been detected inchlorinated disinfectant. A large number of toxicological studies have shown thatiodo-DBPs is stronger cytotoxicity and genetic toxicity compared with chlorinatedand brominated disinfection by-products. In recent years, more and more scholars payattention to the formation and toxicological effect of iodine disinfection by-products.In this paper, we mainly studied the toxic effects of Iodo-DBPs and its relatedmechanisms use HepG-2 cells and CaCo-2 cells, in order to accurately judge thehealth risks to human beings. The results are as follows:1) the morphological changes of iodoacetonitrile on the two kinds of cells wereobserved by inverted microscope. The results showed that the morphology of HepG-2and CaCo-2 cells exposed to 6μM IAN for 24 hours changed obviously. In thetreatment group, the HepG-2 cells were crinkled and round, and the intercellularjunctions were relaxed.In the treatment group, the normal morphology of CaCo-2cells disappeared, the edges of the cells disappeared, and a large number of cellfragments and floating dead cells were observed.The results of MTT assay showedthat all of the eight Iodo-DBPs had an effect on the activity of HepG-2 cells andCaCo-2 cells, and had a significant effect on the change of concentration and time.Theincrease of the exposure concentration or the prolongation of the exposure time willmake the cell proliferation rate decrease obviously.2) The expression levels of reactive oxygen species ( ROS ) , malondialdehyde( MDA ) and total glutathione ( GSH ) in cells were detected. The expression levels ofNRF - 2 and GCLC were detected by RT - PCR, and the expression of NRF-2 andGCLC proteins were detected by Western Blot. The effects of 8 kinds of Iodo - DBPson oxidative damage of cells were investigated . The results showed that all of theeight Iodo-DBPs could induce HepG-2 cells and CaCo-2 cells to produce a large amount of reactive oxygen species (ROS), and the amount of ROS produced wasconsistent with the trend of cytotoxicity. The malondialdehyde (MDA) contentincreased significantly after 8 Iodo-DBPs cells were exposed to HepG-2 cells. Theorder of MDA production of eight Iodo-DBPs exposed to HepG-2 cells was IAA >IAN > BCIM > TIM > BDIM > DBIM > CDIM > DCIM. The content of glutathionein the cells decreased slowly and remained stable with the increase of Iodo-DBPsconcentration. When the concentration of Iodo-DBPs rises to a certain value,Thecontent of GSH in HepG 2 cells also decreased sharply. Iodo-DBPs caused HepG-2cells to initiate the element Nrf-2 to control the redox reaction, and regulated theexpression of downstream gene GCLC in order to maintain a relatively stable state ofredox and precipitate the occurrence of intracellular oxidative damage in HepG-2cells.3) apoptosis rate was detected by flow cytometry and the expression levels ofpro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 were detected by RT-PCR, andthe expression of Bax-Bcl-2caspase-3 protein was detected by Western Blot. Theresults showed that HepG-2 cells and CaCo-2 cells exposed to Iodo-DBPs couldinduce apoptosis. Apoptosis was affected by exposure dose, and the higher theconcentration, the more obvious apoptosis was.The order of apoptosis rate of HepG-2cells was: IAN > IAA > DCIM > CDIM > BCIM > DBIM > TIM > BDIMA CaCo-2cells. The order of apoptosis rate of HepG-2 cells was: IAA > IAN > BDIM >DCIM > DBIM > CDIM > TIM > BCIM. High dose of Iodo-DBPs was the maincause of increasing apoptosis of HepG-2 cells and CaCo-2 cells, and the apoptosisrate of CaCo-2 cells was significantly higher than that of HepG-2 cells. Theexpression of Bax and Bcl-2 in HepG-2 cells was up-regulated 24 hours after acuteexposure to Iodo-DBPs.The Bax/Bcl-2 value was higher than that of the controlgroup.It is concluded that the apoptosis induced by these eight kinds of Iodo-DBPsmay result in the up-regulation of Bax and Caspase-3 protein expression afterexposure to . Iodo-DBPs through mitochondrial pathway, and the increasedconcentration dependence of .Iodo-DBPs exposure may lead to the down-regulationof Bcl-2 protein expression of anti-apoptotic gene.It can be inferred that oxidative stress induced by these eight kinds of Iodo-DBPs may be the cause of inducingapoptosis. |
| Subject Area | 环境科学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14958 |
| Collection | 研究系统_荒漠环境研究室 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 陈娇. 碘代消毒副产物细胞毒性作用及其相关机制研究[D]. 新疆乌鲁木齐. 中国科学院大学,2018. |
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