納米硫化鎘對人腎小管上皮細胞體外毒性研究
發(fā)布時間：2019-02-26 18:56
【摘要】：目的本研究通過體外細胞毒理學(xué)實驗研究納米硫化鎘(nCdS)對人腎小管上皮細胞毒性作用,細胞選擇人腎小管上皮細胞(HKC),探討納米態(tài)硫化鎘對人腎小管上皮細胞(HKC)的氧化損傷過程及其相關(guān)機制。方法1)納米硫化鎘(nCds)實驗材料的表征:分別使用透射電子顯微鏡(TEM)微結(jié)構(gòu)分析和X-射線衍射探傷檢測法(XRD)對實驗所用納米粒子進行表征驗證,達到6~8nm尺度即可用于本研究。2)人腎小管上皮細胞(HKC)的傳代培養(yǎng):將人腎小管上皮細胞(HKC)進行體外傳代培養(yǎng)并對其進行定性鑒定。3)生長情況影響:分別給予HKC不同劑量的納米硫化鎘(nCds),染毒后培養(yǎng)24h,同時設(shè)陰性對照組,采用噻唑藍MTT法(不同劑量組)檢測納米硫化鎘(nCds)對細胞的毒性增值影響。4)細胞形態(tài)觀察:給予HKC不同劑量的納米硫化鎘(nCds)作用24h,同時設(shè)陰性對照組,采用AO/EB染色和DAPI染色后,放置在熒光顯微鏡下觀察HKC形態(tài)的變化,透射電鏡觀察核內(nèi)變化。5)實驗研究納米硫化鎘對HKC內(nèi)氧化應(yīng)激的影響:分別檢測納米硫化鎘處理后的細胞中ROS和GSH含量。6)探討氧化應(yīng)激機制:采用免疫組化法觀察納米硫化鎘對細胞染毒處理后細胞內(nèi)氧化代謝產(chǎn)物丙二醛(MDA)和抗氧化酶包括氧自由基(ROS)、超氧化物歧化酶(SOD)、谷胱甘肽過氧化物還原酶(GSH-Px)、細胞內(nèi)谷胱甘肽(GSH)等物質(zhì)含量變化情況。7)對人腎小管上皮細胞(HKC)內(nèi)炎癥反應(yīng)機制探討:應(yīng)用蛋白免疫印跡法(western blotting法)檢測納米硫化鎘(nCds)染毒處理后的細胞中核轉(zhuǎn)錄因子(NF-κB)蛋白表達水平,間接顯示炎癥反應(yīng)的影響。結(jié)果本研究結(jié)果顯示,隨著實驗組給予人腎小管上皮細胞(HKC)的納米硫化鎘(nCds)染毒處理劑量不斷的增大,細胞存活率呈逐漸降低,與染毒劑量成反比;而生成促氧化物質(zhì)ROS、MDA量升高,抗氧化物質(zhì)GSH-Px、GSH生成量下降,同時觀察到細胞內(nèi)外的Cd2+濃度增加。實驗中將染毒劑量劃分為10.00、20.00、40.00、80.00μg/m L劑量組,實驗隨機分組染毒處理組的細胞存活率隨染毒劑量的增加而下降,其變化具有統(tǒng)計學(xué)意義(P0.05)。結(jié)果顯示各個劑量處理組的ROS和MDA含量以及40.00μg/mL劑量組的GSH與對照組相比較存在差別(P0.05)。隨著納米硫化鎘(nCds)染毒劑量的增加,SOD、ROS、GSH-Px、GSH,40.00μg/m L劑量組的SOD、GSH-Px、GSH的含量下降,ROS、MDA的含量升高,且變化具有統(tǒng)計學(xué)意義(P0.05)。納米硫化鎘(nCdS)對HKC細胞中NF-κB蛋白表達水平的影響,除10μg/m L劑量組未見顯著性差異,其他各個處理組均明與對照組存在差別(P0.05),細胞內(nèi)核轉(zhuǎn)錄因子NF-κB蛋白表達水平隨處理劑量的增加而逐漸升高。結(jié)論1.本研究驗證了納米硫化鎘(nCds)毒性是通過增加活性氧和抑制抗氧化物質(zhì)而誘發(fā)了氧化應(yīng)激反應(yīng),引起線粒體的功能障礙,從而導(dǎo)致不同組織基因表達的改變。2.本研究驗證了納米硫化鎘(nCds)毒性炎癥反應(yīng)是通過核轉(zhuǎn)錄因子NF-κB蛋白等相關(guān)蛋白發(fā)生作用的。3.探討了納米硫化鎘(nCds)毒性機制中氧化應(yīng)激、炎癥反應(yīng)和細胞凋亡相關(guān)路徑的機制。4.納米硫化鎘(nCds)可以引起人腎小管上皮細胞(HKC)的毒性,其機制可能是氧化損傷和Cd2+釋放的共同作用。
[Abstract]:Objective To study the toxicity of nano-sulfide (nCdS) to human renal tubular epithelial cells and to select the human renal tubular epithelial cells (HKC) by in vitro cytotoxicity. The oxidative damage of human renal tubular epithelial cells (HKC) and its related mechanisms were discussed. method 1) the characterization of nano-sulfur (nds) experimental materials: the nano-particles used in the experiment were characterized and verified by using a transmission electron microscope (tem) microstructure analysis and an x-ray diffraction detection method (xrd), respectively, 6-8nm scale was used in this study. 2) Subculture of human renal tubular epithelial cells (HKC): human renal tubular epithelial cells (HKC) were subcultured in vitro and their qualitative identification was carried out. 3) The effect of growth: nano-iron sulfide (nCds) with different doses of HKC, respectively, was given. The effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group) in the negative control group, and the effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group). The changes of the morphology of the HKC were observed with AO/ EB staining and DAPI, and the changes of the morphology of the HKC were observed under the microscope. The content of ROS and GSH in the cells treated with nano-iron sulfide was detected respectively. 6) The mechanism of oxidative stress was discussed: the malondialdehyde (MDA) and the antioxidant enzymes of the oxidized metabolites in the cells after the exposure to the cells were observed by the immunohistochemical method, including the oxygen free radicals (ROS). Changes of the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and intracellular glutathione (GSH) in human renal tubular epithelial cells (HKC) were discussed. The expression level of nuclear transcription factor (NF-EMAB) in the cells treated with nano-iron sulfide (nCds) was detected by Western blotting, and the effect of inflammatory reaction was indirectly shown. Results The results of the study showed that, with the increase of the dose of nano-iron sulfide (nCds) administered to human renal tubular epithelial cells (HKC) in the experimental group, the survival rate of the cells decreased gradually, which was inversely proportional to the exposure dose, and the amount of ROS and MDA was increased. The GSH-Px and GSH content of the anti-oxidant substances decreased, and the Cd2 + concentration inside and outside the cells was observed to increase. In the experiment, the dose group was divided into 10. 00, 20. 00, 40. 00, 80. 000.mu. g/ m L dose group, and the cell survival rate of the experimental group was decreased with the increase of the exposure dose, and the change was statistically significant (P0.05). The results showed that the content of ROS and MDA in each dose treatment group and the content of GSH in the 40. 000.mu. g/ mL dose group were different from the control group (P0.05). The contents of SOD, ROS, GSH-Px, GSH, GSH-Px and GSH decreased, and the content of SOD, GSH-Px and GSH decreased, and the content of ROS and MDA increased with the increase of the dose of nano-iron sulfide (nCds), and the content of ROS and MDA increased, and the change was of statistical significance (P0.05). The effect of nano-sulfide (nCdS) on the expression level of NF-EMAB protein in HKC cells was not found in the 10 & mu; g/ m L dose group, and there was a difference between the other treatment groups and the control group (P0.05). The expression of NF-EMAB protein in the cell core gradually increased with the increase of the treatment dose. Conclusion 1. The present study demonstrated that the toxicity of nano-sulfur (nds) was induced by the increase of active oxygen and the inhibition of antioxidant substances, which led to the dysfunction of the mitochondria, resulting in a change in the expression of different tissue genes. The present study demonstrated that the reaction of nano-sulfur (nCds) toxic inflammation is a function of the related proteins such as the nuclear transcription factor NF-EMAB protein. The mechanisms of oxidative stress, inflammatory response and apoptosis-related pathways in the nCds toxicity mechanism were discussed. Nano-iron sulfide (nCds) can lead to the toxicity of human renal tubular epithelial cells (HKC), and the mechanism may be the co-action of oxidative damage and Cd2 + release.
【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】：2017
【分類號】：R114
本文編號：2431052
[Abstract]:Objective To study the toxicity of nano-sulfide (nCdS) to human renal tubular epithelial cells and to select the human renal tubular epithelial cells (HKC) by in vitro cytotoxicity. The oxidative damage of human renal tubular epithelial cells (HKC) and its related mechanisms were discussed. method 1) the characterization of nano-sulfur (nds) experimental materials: the nano-particles used in the experiment were characterized and verified by using a transmission electron microscope (tem) microstructure analysis and an x-ray diffraction detection method (xrd), respectively, 6-8nm scale was used in this study. 2) Subculture of human renal tubular epithelial cells (HKC): human renal tubular epithelial cells (HKC) were subcultured in vitro and their qualitative identification was carried out. 3) The effect of growth: nano-iron sulfide (nCds) with different doses of HKC, respectively, was given. The effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group) in the negative control group, and the effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group). The changes of the morphology of the HKC were observed with AO/ EB staining and DAPI, and the changes of the morphology of the HKC were observed under the microscope. The content of ROS and GSH in the cells treated with nano-iron sulfide was detected respectively. 6) The mechanism of oxidative stress was discussed: the malondialdehyde (MDA) and the antioxidant enzymes of the oxidized metabolites in the cells after the exposure to the cells were observed by the immunohistochemical method, including the oxygen free radicals (ROS). Changes of the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and intracellular glutathione (GSH) in human renal tubular epithelial cells (HKC) were discussed. The expression level of nuclear transcription factor (NF-EMAB) in the cells treated with nano-iron sulfide (nCds) was detected by Western blotting, and the effect of inflammatory reaction was indirectly shown. Results The results of the study showed that, with the increase of the dose of nano-iron sulfide (nCds) administered to human renal tubular epithelial cells (HKC) in the experimental group, the survival rate of the cells decreased gradually, which was inversely proportional to the exposure dose, and the amount of ROS and MDA was increased. The GSH-Px and GSH content of the anti-oxidant substances decreased, and the Cd2 + concentration inside and outside the cells was observed to increase. In the experiment, the dose group was divided into 10. 00, 20. 00, 40. 00, 80. 000.mu. g/ m L dose group, and the cell survival rate of the experimental group was decreased with the increase of the exposure dose, and the change was statistically significant (P0.05). The results showed that the content of ROS and MDA in each dose treatment group and the content of GSH in the 40. 000.mu. g/ mL dose group were different from the control group (P0.05). The contents of SOD, ROS, GSH-Px, GSH, GSH-Px and GSH decreased, and the content of SOD, GSH-Px and GSH decreased, and the content of ROS and MDA increased with the increase of the dose of nano-iron sulfide (nCds), and the content of ROS and MDA increased, and the change was of statistical significance (P0.05). The effect of nano-sulfide (nCdS) on the expression level of NF-EMAB protein in HKC cells was not found in the 10 & mu; g/ m L dose group, and there was a difference between the other treatment groups and the control group (P0.05). The expression of NF-EMAB protein in the cell core gradually increased with the increase of the treatment dose. Conclusion 1. The present study demonstrated that the toxicity of nano-sulfur (nds) was induced by the increase of active oxygen and the inhibition of antioxidant substances, which led to the dysfunction of the mitochondria, resulting in a change in the expression of different tissue genes. The present study demonstrated that the reaction of nano-sulfur (nCds) toxic inflammation is a function of the related proteins such as the nuclear transcription factor NF-EMAB protein. The mechanisms of oxidative stress, inflammatory response and apoptosis-related pathways in the nCds toxicity mechanism were discussed. Nano-iron sulfide (nCds) can lead to the toxicity of human renal tubular epithelial cells (HKC), and the mechanism may be the co-action of oxidative damage and Cd2 + release.
【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】：2017
【分類號】：R114
【參考文獻】
相關(guān)期刊論文 前10條
1 李津;王曉軍;張靜姝;王鳳山;何寧;劉英華;;納米硫化鎘對人腎小管上皮細胞氧化損傷的影響[J];職業(yè)與健康;2015年24期
2 劉明閣;宋魏;張建波;孫淼淼;于慶凱;;NF-κB在B細胞淋巴瘤中的表達和意義[J];河南醫(yī)學(xué)研究;2015年01期
3 侯俊;代珊珊;王超;王沛芳;苗令占;許伊;;人工納米材料與生物膜相互作用機制及影響因素研究進展[J];微生物學(xué)通報;2015年02期
4 周慶紅;姜淑卿;劉英華;張力;趙春紅;王曉軍;張大龍;張靜姝;;納米硫化鎘與常規(guī)硫化鎘對雄性小鼠的生殖毒性[J];環(huán)境與健康雜志;2014年04期
5 王曉軍;張靜姝;何寧;劉英華;周慶紅;張大龍;商博東;李津;;納米硫化鎘對人腎小球系膜細胞氧化損傷及NF-κB蛋白表達的影響[J];環(huán)境與健康雜志;2014年04期
6 張靜;王曉軍;張靜姝;姜淑卿;李敏;何寧;;納米硫化鎘對人腎小球系膜細胞的氧化損傷研究[J];環(huán)境與健康雜志;2014年03期
7 王蓉;林溢琦;孫相振;鐘秋;耿輝;熊麗;;納米硫化鎘量子點對斜生柵藻的毒性研究[J];農(nóng)業(yè)環(huán)境科學(xué)學(xué)報;2013年03期
8 周海英;周瑞;熊斌;何彥;;大長徑比金納米棒的合成及其單細胞毒性研究[J];分析化學(xué);2012年12期
9 王煜倩;賈光;沈臻霖;張杰;唐仕川;張斌;;納米二氧化鈦對肺部損傷研究進展[J];中國安全生產(chǎn)科學(xué)技術(shù);2012年04期
10 趙倩;李興;;脂聯(lián)素對高糖環(huán)境下人腎小球系膜細胞增殖及氧化應(yīng)激水平的影響[J];中國現(xiàn)代藥物應(yīng)用;2012年08期
，本文編號：2431052
本文鏈接：http://sikaile.net/yixuelunwen/yufangyixuelunwen/2431052.html
最近更新
教材專著
