微囊藻毒素-LR對(duì)小鼠肝細(xì)胞的氧化損傷及γ-GCS基因表達(dá)的影響
發(fā)布時(shí)間：2018-08-01 08:25
【摘要】：目的：研究微囊藻毒素-LR（Microcystin-LR，MC-LR）短期重復(fù)暴露對(duì)小鼠肝細(xì)胞產(chǎn)生的氧化損傷效應(yīng)，以及對(duì)γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthethase,γ-GCS)相關(guān)亞基的mRNA、蛋白表達(dá)水平的影響，探討MC-LR誘導(dǎo)的肝細(xì)胞氧化損傷與γ-GCS基因表達(dá)水平之間的關(guān)系。 方法：80只健康昆明小鼠，隨機(jī)分為2批，每批4組，每組10只，分別為對(duì)照組（含0.02%二甲基亞砜的生理鹽水0.005ml/g）、低劑量組（MC-LR5μg/kg）、中劑量組（MC-LR10μg/kg）、高劑量組（MC-LR20μg/kg），分別染毒10天和20天。每日1次經(jīng)腹腔注射染毒，每隔1日稱量體重，觀察小鼠體重增長(zhǎng)情況，計(jì)算體重增長(zhǎng)率。分別于染毒第11天和第21天處死小鼠，取肝臟組織。用硫代巴比妥酸（TBA）法測(cè)定丙二醛（malondialdehyde，MDA）含量，用二硫代硝基苯甲酸（DTNB）法測(cè)定GSH含量；用免疫組織化學(xué)法測(cè)定DNA氧化損傷標(biāo)志物8-羥基脫氧鳥(niǎo)苷（8-hydroxydeoxiguanosine，8-OHdG）水平；提取肝細(xì)胞RNA，逆轉(zhuǎn)錄成cDNA，用Real-time PCR法測(cè)定γ-GCS催化亞基(Glutamate cysteine ligasecatalytic subunit,GCLC)和調(diào)節(jié)亞基（Glutamate cysteine ligase modifiersubunit，GCLM）的mRNA表達(dá)水平；提取肝細(xì)胞蛋白用蛋白免疫印跡法（Western blot，WB）測(cè)定GCLC和GCLM的蛋白表達(dá)水平。 結(jié)果：1、經(jīng)10天和20天染毒后，低劑量組小鼠的體重增長(zhǎng)率與對(duì)照組比較沒(méi)有明顯差異（P0.05），中、高劑量組小鼠體重增長(zhǎng)率均顯著低于其對(duì)照組，，差異有統(tǒng)計(jì)學(xué)意義（P0.05），且體重增長(zhǎng)率均隨染毒劑量的增加而降低。 2、MC-LR染毒10天和20天后，低劑量染毒對(duì)小鼠肝組織MDA水平影響不明顯，差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05），而中、高劑量組MDA水平明顯高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 3、染毒10天后，低劑量組小鼠肝組織GSH含量與對(duì)照組比較無(wú)明顯差異（P0.05），中、高劑量組GSH水平低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；染毒20天后，各劑量組GSH水平均低于對(duì)照組，有顯著性差異（P0.05）。 4、染毒10天和20天后，低、中、高劑量組小鼠肝細(xì)胞8-OHdG水平均高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P0.05)，且隨著MC-LR染毒劑量的增加上升。 5、MC-LR染毒10天后，低、中、高劑量組肝細(xì)胞GCLC mRNA表達(dá)水平分別降低至對(duì)照組的72.0%、44.1%和46.5%，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）；MC-LR染毒20天后，各劑量組GCLC mRNA表達(dá)水平分別低至對(duì)照組的68.5%、26.6%和26.7%，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。 6、MC-LR染毒10天后，低、中、高劑量組小鼠肝細(xì)胞GCLM mRNA表達(dá)水平明顯低于對(duì)照組，分別降至對(duì)照組的59.4%、42.5%和34.3%，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；染毒20天結(jié)果顯示，低、中、高劑量組GCLMmRNA表達(dá)水平分別降低至對(duì)照組的62.9%、35.8%和39.0%（P0.05）。 7、MC-LR染毒10天及20天，低劑量組小鼠肝細(xì)胞GCLC蛋白表達(dá)量均無(wú)顯著性變化（P0.05），但中、高劑量組GCLC蛋白表達(dá)量均明顯低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；染毒20天后，低、中、高劑量組GCLC蛋白表達(dá)量均低于染毒10天相應(yīng)的劑量組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 8、染毒10天后，低劑量組小鼠肝細(xì)胞GCLM蛋白表達(dá)量與對(duì)照組比較無(wú)顯著性差異，中、高劑量組GCLM蛋白表達(dá)水平均明顯低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；染毒20天后，低、中、高劑量組GCLM蛋白表達(dá)水平均低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；中劑量組染毒20天后，GCLM蛋白表達(dá)水平明顯低于染毒10天的水平，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 9、肝細(xì)胞GCLC、GCLM mRNA表達(dá)水平與MDA水平呈正相關(guān)關(guān)系（rGCLC=0.425，P0.05；rGCLM=0.304，P0.05）；肝細(xì)胞GCLC、GCLM mRNA表達(dá)水平與8-OHdG水平呈正相關(guān)關(guān)系（rGCLC=0.420，P0.05；rGCLM=0.476，P0.05）。結(jié)論： MC-LR可抑制小鼠肝細(xì)胞γ-GCS的催化亞基GCLC、調(diào)節(jié)亞基GLCM的mRNA表達(dá)和蛋白表達(dá)，降低GSH水平，升高M(jìn)DA和8-OHdG水平，引起脂質(zhì)和DNA氧化損傷，但MC-LR對(duì)γ-GCS表達(dá)的影響機(jī)制仍有待于進(jìn)一步研究。
[Abstract]:Objective: To study the oxidative damage effect of microcystin -LR (Microcystin-LR, MC-LR) on mice liver cells, and the effect on the mRNA and protein expression level of gamma glutamyl cysteine synthetase (gamma -glutamylcysteine synthethase, gamma -GCS) related subunits, and to explore the oxidative damage of hepatocytes and gamma -GCS induced by MC-LR. The relationship between the level of gene expression.
Methods: 80 healthy Kunming mice were randomly divided into 2 batches, each group of 4 groups, 10 in each group, the control group (including 0.02% two methyl sulfoxide 0.005ml/g), the low dose group (MC-LR5 mu g/kg), the medium dose group (MC-LR10 mu g/kg), the high dose group (MC-LR20 mu g/kg), respectively for 10 days and 20 days, 1 times a day by intraperitoneal injection, and every 1 days. The weight growth of mice was observed and the growth rate of body weight was calculated. The mice were killed for eleventh days and twenty-first days and the liver tissues were taken respectively. The content of malondialdehyde (malondialdehyde, MDA) was determined by thiobarbituric acid (TBA) method and the content of GSH was measured with two thiobenzoic acid (DTNB) method. The oxidative damage standard of DNA was measured by immunohistochemistry. The level of 8- hydroxy deoxy guanosine (8-hydroxydeoxiguanosine, 8-OHdG) was extracted, RNA of liver cells was extracted and cDNA was reverse transcriptase, and the expression level of gamma -GCS catalytic subunit (Glutamate cysteine ligasecatalytic subunit) and regulating subunit was extracted by Real-time PCR method; the liver cell eggs were extracted. The protein expression levels of GCLC and GCLM were measured by Western blot (WB).
Results: 1, after 10 days and 20 days of poisoning, the weight growth rate of mice in low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the weight growth rate of the mice was significantly lower than that of the control group, the difference was statistically significant (P0.05), and the weight growth rate decreased with the increase of the dose.
2, after 10 and 20 days of MC-LR poisoning, the effect of low dose on the MDA level of liver tissue in mice was not significant (P0.05), but in the high dose group, the level of MDA was significantly higher than that in the control group, the difference was statistically significant (P0.05).
3, after 10 days of poisoning, the GSH content in the liver tissue of the low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the GSH level was lower than the control group, the difference was statistically significant (P0.05), and the GSH level in each dose group was lower than that of the control group for 20 days, and there was a significant difference (P0.05).
4, after 10 and 20 days, the levels of 8-OHdG in the liver cells of the low, middle and high dose groups were all higher than those of the control group, the difference was statistically significant (P0.05), and increased with the increase of the dose of MC-LR.
5, after 10 days of MC-LR poisoning, the expression level of GCLC mRNA in liver cells in low, middle and high dose groups decreased to 72%, 44.1% and 46.5% in the control group, and the difference was statistically significant (P0.05). After 20 days of MC-LR, the expression level of GCLC mRNA in each dose group was 68.5%, 26.6% and 26.7% in the control group respectively. The difference was statistically significant (P0.05).
6, after 10 days of MC-LR poisoning, the expression level of GCLM mRNA in the hepatocytes of low, middle and high dose mice was significantly lower than that of the control group, which decreased to 59.4%, 42.5% and 34.3% of the control group, respectively (P0.05). The GCLMmRNA expression level in the low, middle and high dose groups decreased to 62.9%, 35.8% and 39% (P0.05) in the lower, middle and high dose groups respectively.
7, MC-LR was poisoned for 10 days and 20 days, and there was no significant change in the expression of GCLC protein in the liver cells of the low dose group (P0.05), but the expression of GCLC protein in the high dose group was significantly lower than that of the control group. The difference was statistically significant (P0.05). The expression of GCLC protein in the low, middle and high dose group was lower than that of the corresponding dose group of the 10 day poisoned group, and the difference was lower than that of the corresponding dose group for 10 days. There was a statistical significance (P0.05).
8, after 10 days of poisoning, there was no significant difference in the expression of GCLM protein in the liver cells of the low dose group with the control group, and the expression level of GCLM protein in the high dose group was significantly lower than that of the control group (P0.05), and the expression level of GCLM protein in the low, middle and high dose group was lower than that of the control group for 20 days, and the difference was statistically significant (P The expression of GCLM protein in middle dose group was significantly lower than that in 10 days after exposure (P 0.05).
9, the expression level of GCLC and GCLM mRNA was positively correlated with the level of MDA (rGCLC=0.425, P0.05; rGCLM=0.304, P0.05), and the expression level of the hepatocyte GCLC and GCLM mRNA was positively correlated with the 8-OHdG level. The expression of mRNA and protein, decrease the level of GSH and increase the level of MDA and 8-OHdG, cause the oxidative damage of lipid and DNA, but the mechanism of MC-LR on the expression of gamma -GCS remains to be further studied.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575
本文編號(hào)：2156968
[Abstract]:Objective: To study the oxidative damage effect of microcystin -LR (Microcystin-LR, MC-LR) on mice liver cells, and the effect on the mRNA and protein expression level of gamma glutamyl cysteine synthetase (gamma -glutamylcysteine synthethase, gamma -GCS) related subunits, and to explore the oxidative damage of hepatocytes and gamma -GCS induced by MC-LR. The relationship between the level of gene expression.
Methods: 80 healthy Kunming mice were randomly divided into 2 batches, each group of 4 groups, 10 in each group, the control group (including 0.02% two methyl sulfoxide 0.005ml/g), the low dose group (MC-LR5 mu g/kg), the medium dose group (MC-LR10 mu g/kg), the high dose group (MC-LR20 mu g/kg), respectively for 10 days and 20 days, 1 times a day by intraperitoneal injection, and every 1 days. The weight growth of mice was observed and the growth rate of body weight was calculated. The mice were killed for eleventh days and twenty-first days and the liver tissues were taken respectively. The content of malondialdehyde (malondialdehyde, MDA) was determined by thiobarbituric acid (TBA) method and the content of GSH was measured with two thiobenzoic acid (DTNB) method. The oxidative damage standard of DNA was measured by immunohistochemistry. The level of 8- hydroxy deoxy guanosine (8-hydroxydeoxiguanosine, 8-OHdG) was extracted, RNA of liver cells was extracted and cDNA was reverse transcriptase, and the expression level of gamma -GCS catalytic subunit (Glutamate cysteine ligasecatalytic subunit) and regulating subunit was extracted by Real-time PCR method; the liver cell eggs were extracted. The protein expression levels of GCLC and GCLM were measured by Western blot (WB).
Results: 1, after 10 days and 20 days of poisoning, the weight growth rate of mice in low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the weight growth rate of the mice was significantly lower than that of the control group, the difference was statistically significant (P0.05), and the weight growth rate decreased with the increase of the dose.
2, after 10 and 20 days of MC-LR poisoning, the effect of low dose on the MDA level of liver tissue in mice was not significant (P0.05), but in the high dose group, the level of MDA was significantly higher than that in the control group, the difference was statistically significant (P0.05).
3, after 10 days of poisoning, the GSH content in the liver tissue of the low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the GSH level was lower than the control group, the difference was statistically significant (P0.05), and the GSH level in each dose group was lower than that of the control group for 20 days, and there was a significant difference (P0.05).
4, after 10 and 20 days, the levels of 8-OHdG in the liver cells of the low, middle and high dose groups were all higher than those of the control group, the difference was statistically significant (P0.05), and increased with the increase of the dose of MC-LR.
5, after 10 days of MC-LR poisoning, the expression level of GCLC mRNA in liver cells in low, middle and high dose groups decreased to 72%, 44.1% and 46.5% in the control group, and the difference was statistically significant (P0.05). After 20 days of MC-LR, the expression level of GCLC mRNA in each dose group was 68.5%, 26.6% and 26.7% in the control group respectively. The difference was statistically significant (P0.05).
6, after 10 days of MC-LR poisoning, the expression level of GCLM mRNA in the hepatocytes of low, middle and high dose mice was significantly lower than that of the control group, which decreased to 59.4%, 42.5% and 34.3% of the control group, respectively (P0.05). The GCLMmRNA expression level in the low, middle and high dose groups decreased to 62.9%, 35.8% and 39% (P0.05) in the lower, middle and high dose groups respectively.
7, MC-LR was poisoned for 10 days and 20 days, and there was no significant change in the expression of GCLC protein in the liver cells of the low dose group (P0.05), but the expression of GCLC protein in the high dose group was significantly lower than that of the control group. The difference was statistically significant (P0.05). The expression of GCLC protein in the low, middle and high dose group was lower than that of the corresponding dose group of the 10 day poisoned group, and the difference was lower than that of the corresponding dose group for 10 days. There was a statistical significance (P0.05).
8, after 10 days of poisoning, there was no significant difference in the expression of GCLM protein in the liver cells of the low dose group with the control group, and the expression level of GCLM protein in the high dose group was significantly lower than that of the control group (P0.05), and the expression level of GCLM protein in the low, middle and high dose group was lower than that of the control group for 20 days, and the difference was statistically significant (P The expression of GCLM protein in middle dose group was significantly lower than that in 10 days after exposure (P 0.05).
9, the expression level of GCLC and GCLM mRNA was positively correlated with the level of MDA (rGCLC=0.425, P0.05; rGCLM=0.304, P0.05), and the expression level of the hepatocyte GCLC and GCLM mRNA was positively correlated with the 8-OHdG level. The expression of mRNA and protein, decrease the level of GSH and increase the level of MDA and 8-OHdG, cause the oxidative damage of lipid and DNA, but the mechanism of MC-LR on the expression of gamma -GCS remains to be further studied.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 許川;舒為群;曹佳;;我國(guó)水環(huán)境微囊藻毒素污染及其健康危害研究[J];癌變.畸變.突變;2007年03期
2 丁笑生;;微囊藻毒素及其腎毒性的研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2008年31期
3 曹守冬;谷氨酰半胱氨酸合成酶調(diào)節(jié)機(jī)制研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè));2003年01期
4 農(nóng)清清;竹內(nèi)亨;張志勇;何敏;Masahisa Horiuchi;;谷胱甘肽耗竭與微囊藻毒素-LR細(xì)胞毒性的關(guān)系初探[J];應(yīng)用預(yù)防醫(yī)學(xué);2008年03期
5 董丹丹;王欣;趙朔;段曉旭;李煒;邢曉越;李冰;;急性砷暴露對(duì)小鼠機(jī)體谷胱甘肽水平及其調(diào)控酶蛋白表達(dá)的影響[J];環(huán)境與健康雜志;2013年01期
6 高艷芳;郭有;范興君;王伯超;胡小山;胡姿浩;胡恭華;;低劑量微囊藻毒素對(duì)小鼠肝功能及氧化損傷的影響[J];實(shí)用醫(yī)學(xué)雜志;2012年15期
7 吳和巖,鄭力行,蘇瑾,施瑋;上海市供水系統(tǒng)微囊藻毒素LR含量調(diào)查[J];衛(wèi)生研究;2005年02期
8 程磷令;γ-谷氨酰半胱氨酸合成酶基因及其調(diào)控[J];國(guó)外醫(yī)學(xué)(生理、病理科學(xué)與臨床分冊(cè));2002年06期
9 陳鐵暉;薛常鎬;汪家梨;謝建忠;曾毅丹;陳華;;姜黃素拮抗水華微囊藻毒素致動(dòng)物肝氧化損傷[J];中國(guó)公共衛(wèi)生;2006年11期
10 王偉琴;金永堂;吳斌;孫肖瑜;龐曉露;王靜;;水源水中微囊藻毒素的遺傳毒性與健康風(fēng)險(xiǎn)評(píng)價(jià)[J];中國(guó)環(huán)境科學(xué);2010年04期
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