微囊藻毒素-LR对小鼠肝细胞的氧化损伤及γ-GCS基因表达的影响

发布时间：2018-08-01 08:25

【摘要】：目的：研究微囊藻毒素-LR（Microcystin-LR，MC-LR）短期重复暴露对小鼠肝细胞产生的氧化损伤效应，以及对γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthethase,γ-GCS)相关亚基的mRNA、蛋白表达水平的影响，探讨MC-LR诱导的肝细胞氧化损伤与γ-GCS基因表达水平之间的关系。方法：80只健康昆明小鼠，随机分为2批，每批4组，每组10只，分别为对照组（含0.02%二甲基亚砜的生理盐水0.005ml/g）、低剂量组（MC-LR5μg/kg）、中剂量组（MC-LR10μg/kg）、高剂量组（MC-LR20μg/kg），分别染毒10天和20天。每日1次经腹腔注射染毒，每隔1日称量体重，观察小鼠体重增长情况，计算体重增长率。分别于染毒第11天和第21天处死小鼠，取肝脏组织。用硫代巴比妥酸（TBA）法测定丙二醛（malondialdehyde，MDA）含量，用二硫代硝基苯甲酸（DTNB）法测定GSH含量；用免疫组织化学法测定DNA氧化损伤标志物8-羟基脱氧鸟苷（8-hydroxydeoxiguanosine，8-OHdG）水平；提取肝细胞RNA，逆转录成cDNA，用Real-time PCR法测定γ-GCS催化亚基(Glutamate cysteine ligasecatalytic subunit,GCLC)和调节亚基（Glutamate cysteine ligase modifiersubunit，GCLM）的mRNA表达水平；提取肝细胞蛋白用蛋白免疫印迹法（Western blot，WB）测定GCLC和GCLM的蛋白表达水平。结果：1、经10天和20天染毒后，低剂量组小鼠的体重增长率与对照组比较没有明显差异（P0.05），中、高剂量组小鼠体重增长率均显著低于其对照组，，差异有统计学意义（P0.05），且体重增长率均随染毒剂量的增加而降低。 2、MC-LR染毒10天和20天后，低剂量染毒对小鼠肝组织MDA水平影响不明显，差异无统计学意义（P0.05），而中、高剂量组MDA水平明显高于对照组，差异有统计学意义（P0.05）。 3、染毒10天后，低剂量组小鼠肝组织GSH含量与对照组比较无明显差异（P0.05），中、高剂量组GSH水平低于对照组，差异有统计学意义（P0.05）；染毒20天后，各剂量组GSH水平均低于对照组，有显著性差异（P0.05）。 4、染毒10天和20天后，低、中、高剂量组小鼠肝细胞8-OHdG水平均高于对照组，差异有统计学意义(P0.05)，且随着MC-LR染毒剂量的增加上升。 5、MC-LR染毒10天后，低、中、高剂量组肝细胞GCLC mRNA表达水平分别降低至对照组的72.0%、44.1%和46.5%，差异均有统计学意义（P0.05）；MC-LR染毒20天后，各剂量组GCLC mRNA表达水平分别低至对照组的68.5%、26.6%和26.7%，差异均有统计学意义（P0.05）。 6、MC-LR染毒10天后，低、中、高剂量组小鼠肝细胞GCLM mRNA表达水平明显低于对照组，分别降至对照组的59.4%、42.5%和34.3%，差异有统计学意义（P0.05）；染毒20天结果显示，低、中、高剂量组GCLMmRNA表达水平分别降低至对照组的62.9%、35.8%和39.0%（P0.05）。 7、MC-LR染毒10天及20天，低剂量组小鼠肝细胞GCLC蛋白表达量均无显著性变化（P0.05），但中、高剂量组GCLC蛋白表达量均明显低于对照组，差异有统计学意义（P0.05）；染毒20天后，低、中、高剂量组GCLC蛋白表达量均低于染毒10天相应的剂量组，差异有统计学意义（P0.05）。 8、染毒10天后，低剂量组小鼠肝细胞GCLM蛋白表达量与对照组比较无显著性差异，中、高剂量组GCLM蛋白表达水平均明显低于对照组，差异有统计学意义（P0.05）；染毒20天后，低、中、高剂量组GCLM蛋白表达水平均低于对照组，差异有统计学意义（P0.05）；中剂量组染毒20天后，GCLM蛋白表达水平明显低于染毒10天的水平，差异有统计学意义（P0.05）。 9、肝细胞GCLC、GCLM mRNA表达水平与MDA水平呈正相关关系（rGCLC=0.425，P0.05；rGCLM=0.304，P0.05）；肝细胞GCLC、GCLM mRNA表达水平与8-OHdG水平呈正相关关系（rGCLC=0.420，P0.05；rGCLM=0.476，P0.05）。结论： MC-LR可抑制小鼠肝细胞γ-GCS的催化亚基GCLC、调节亚基GLCM的mRNA表达和蛋白表达，降低GSH水平，升高MDA和8-OHdG水平，引起脂质和DNA氧化损伤，但MC-LR对γ-GCS表达的影响机制仍有待于进一步研究。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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