本文選題：微囊藻毒素-LR + 動物實驗��； 參考：《華中科技大學》2013年博士論文

【摘要】：第一部分微囊藻毒素-LR暴露生物標志物的研究 目的：通過檢測動物血液和組織中微囊藻毒素-LR單體及復(fù)合物，結(jié)合篩查血液細胞可能發(fā)生的形態(tài)、功能和分子生物學的變化及血液生化指標改變，初步確立能夠反映機體暴露藻類毒素污染的生物負荷和生物效應(yīng)指標，為環(huán)境藻類毒素污染與健康評價提供科學實驗方法和手段。 方法：SPF級雄性昆明小鼠，體重在20~25g之間，用隨機數(shù)字表法分為5組，每組7只。對照組、低劑量組、中低劑量組、中高劑量組、高劑量組，分別以生理鹽水、3.125、6.250、12.500、25.000μg/(kg·d)劑量微囊藻毒素-LR給予腹腔注射染毒。染毒7天后，進行血液生化和血液細胞分析，檢測血清細胞因子， KCl-SDS沉淀法檢測淋巴細胞DNA-蛋白質(zhì)交聯(lián)，流式細胞儀檢測單核細胞吞噬功能及單核細胞和淋巴細胞的活性氧，，高效液相色譜-質(zhì)譜聯(lián)用技術(shù)檢測血液及組織中的微囊藻毒素-LR，并分析其變化情況。 結(jié)果：本研究中各項指標對于微囊藻毒素-LR暴露的敏感度從高至低依次為：肝臟中檢測到的微囊藻毒素-LR，巨噬細胞吞噬功能和白細胞活性氧；白細胞介素-6，堿性磷酸酶和乳酸脫氫酶；丙氨酸氨基轉(zhuǎn)移酶和血液中檢測到的微囊藻毒素-LR；天門冬氨酸氨基轉(zhuǎn)移酶和腫瘤壞死因子-α。3.125、6.250、12.500、25.000μg/(kg·d)染毒組肝臟中均檢測到微囊藻毒素-LR（分別為0.1529±0.0296，0.3047±0.0648，0.6006±0.1105，0.9899±0.2261μg/g肝臟）。同樣劑量染毒組淋巴細胞DCF熒光強度（依次為3299.37±120.54，3281.38±58.34，3308.06±136.12，3346.92±108.69）均低于對照組（3770.81±131.39）（P 0.05）；單核細胞DCF熒光強度（依次為3271.51±140.79，3270.05±117.92，3326.90±114.39，3292.49±145.97）均低于對照組（3841.72±130.92）（P 0.05）。6.250、12.500、25.000μg/(kg·d)染毒組小鼠白細胞介素-6（分別為346.837±25.536，360.847±37.886，434.245±35.858pg/mL）均高于對照組（232.775±32.816pg/mL）（P 0.05）；而只有最高劑量25.000μg/(kg·d)染毒組腫瘤壞死因子-α（10.782±0.966fmol/mL）低于對照組（16.878±3.378fmol/mL）（P 0.05）。血清堿性磷酸酶和乳酸脫氫酶在6.250μg/(kg·d)即高于陰性對照組，差異有統(tǒng)計學意義（P 0.05）。 結(jié)論：本研究對今后的流行病學調(diào)查有一定的參考價值，血液和肝臟中檢測到微囊藻毒素-LR是暴露的確證標志物，結(jié)合血液生化分析、活性氧、細胞因子等的改變可綜合分析機體的暴露情況，為微囊藻毒素的研究提供更詳細全面的資料。 第二部分微囊藻毒素-LR對肝細胞凋亡及Caspase-3、凋亡誘導(dǎo)因子、核酸內(nèi)切酶G mRNA表達的影響 目的：通過觀察微囊藻毒素-LR對肝細胞凋亡及Caspase-3、凋亡誘導(dǎo)因子、核酸內(nèi)切酶G mRNA表達的影響，探討微囊藻毒素-LR對肝細胞凋亡的影響，豐富微囊藻毒素-LR的凋亡誘導(dǎo)機制。 方法：SPF級雄性昆明小鼠，體重在20~25g之間，用隨機數(shù)字表法分為5組，每組7只。對照組、低劑量組、中低劑量組、中高劑量組、高劑量組，分別以生理鹽水、3.125、6.250、12.500、25.000μg/(kg·d)劑量微囊藻毒素-LR給予腹腔注射染毒。染毒7天后，HE染色觀察肝臟病理變化，TUNEL染色觀察肝細胞凋亡情況，熒光定量PCR檢測Caspase-3、凋亡誘導(dǎo)因子、核酸內(nèi)切酶G mRNA表達變化。 結(jié)果：小劑量微囊藻毒素-LR暴露導(dǎo)致肝臟細胞出現(xiàn)較輕的損傷和嚴重的凋亡；隨著染毒劑量增加，肝細胞損傷逐漸加重，但肝細胞凋亡并未隨染毒劑量升高而增加。3.125、6.250、12.500、25.000μg/(kg·d)微囊藻毒素-LR染毒組肝臟組織細胞凋亡指數(shù)（分別為46.51±4.69，11.08±3.18，10.72±3.36，10.44±3.48）均高于陰性對照組（3.00±0.60）（P 0.05）。隨著微囊藻毒素-LR染毒劑量的增加，肝細胞Caspase-3、凋亡誘導(dǎo)因子mRNA表達下降；3.125、6.250、12.500、25.000μg/(kg·d)染毒組Caspase-3mRNA表達（分別為0.8214±0.1150，0.5594±0.1800，0.3842±0.0745，0.3158±0.1307）均低于陰性對照組（1.000）（P 0.05）。本研究微囊藻毒素-LR染毒劑量不影響肝細胞核酸內(nèi)切酶G mRNA表達。 結(jié)論：微囊藻毒素-LR的促腫瘤作用可能與Caspase-3、凋亡誘導(dǎo)因子mRNA表達異常有關(guān)。
[Abstract]:Part one studies on biomarkers of microcystin -LR exposure
Objective: by detecting the microcystin -LR monomer and complex in animal blood and tissue, combining the possible morphological changes of blood cells, the changes in function and molecular biology and the change of blood biochemical indexes, the biological effects and biological effects of algae toxin pollution in the body were preliminarily established. Provide scientific experimental methods and means for pollution and health evaluation.
Methods: SPF male Kunming mice, with a weight of 20~25g, were divided into 5 groups, with 7 rats in each group. The control group, low dose group, middle and low dose group, middle and high dose group, high dose group, 3.125,6.250,12.500,25.000 mu g/ (kg. D) dose microcystin -LR were given intraperitoneal injection respectively. After 7 days of poisoning, blood was carried out. Serum cytokine, serum cytokine, serum cytokine, KCl-SDS precipitation, DNA- protein crosslinking, phagocytosis of mononuclear cells and mononuclear cells and lymphocytes were detected by flow cytometry, and microcystin -LR in blood and tissue was detected by high performance liquid chromatography-mass spectrometry (HPLC MS), and its changes were analyzed. Change the situation.
Results: the sensitivity of each index to microcystin -LR exposure from high to low was microcystin -LR, macrophage phagocytosis and leucocyte reactive oxygen species, interleukin -6, alkaline phosphatase and lactate dehydrogenase, alanine aminotransferase and Microcystis detected in blood Toxin -LR, microcystin -LR (0.1529 + 0.0296,0.3047 + 0.0648,0.6006 + 0.2261 micron g/g liver respectively) was detected in the liver of aspartic aminotransferase and tumor necrosis factor - alpha.3.125,6.250,12.500,25.000 g/ (kg. D). The DCF fluorescence intensity of the lymphocytes in the same dose group was 3299.37, respectively. The 120.543281.38 + 58.343308.06 + 136.123346.92 + 108.69) was lower than that of the control group (3770.81 + 131.39) (P 0.05), and the DCF fluorescence intensity of mononuclear cells (3271.51 + 140.793270.05 + 114.393292.49 + 145.97) was lower than that of the control group (3841.72 + 130.92) (P 0.05).6.250,12.500,25.000 mu g/ (kg. The cytokine -6 (346.837 + 25.536360.847 + 37.886434.245 + 35.858pg/mL respectively) was higher than that of the control group (232.775 + 32.816pg/mL) (P 0.05), but only the highest dose 25 mu g/ (kg. D) tumor necrosis factor - alpha (10.782 + 0.966fmol/mL) was lower than the control group (16.878 + 3.378fmol/mL) (P 0.05). Serum alkaline phosphatase and lactate dehydrogenation The enzyme was higher than the negative control group at 6.250 g/ (kg. D), the difference was statistically significant (P 0.05).
Conclusion: This study has a certain reference value for future epidemiological investigation. Microcystin -LR in the blood and liver is a confirmatory marker of exposure. The changes of blood biochemical analysis, reactive oxygen species and cytokine can be used to analyze the exposure of the body and provide more detailed and comprehensive information for the study of microcystin.
The second part is the effect of microcystin -LR on hepatocyte apoptosis and expression of Caspase-3, apoptosis inducing factor and endonuclease G mRNA.
Objective: To investigate the effect of microcystin -LR on hepatocyte apoptosis and the expression of Caspase-3, apoptosis inducible factor and endonuclease G mRNA, and to explore the effect of microcystin -LR on hepatocyte apoptosis and to enrich the apoptosis induction mechanism of microcystin -LR.
Methods: SPF male Kunming mice, with a weight of 20~25g, were divided into 5 groups, with 7 rats in each group. The control group, low dose group, middle and low dose group, middle and high dose group, high dose group, 3.125,6.250,12.500,25.000 mu g/ (kg. D) dose microcystin -LR were given intraperitoneal injection respectively. After 7 days of poisoning, HE staining concept was used. The pathological changes of liver were observed. The apoptosis of hepatocytes was observed by TUNEL staining. The expression of Caspase-3, apoptosis inducing factor and endonuclease G mRNA were detected by fluorescent quantitative PCR.
Results: the exposure of small dose of microcystin -LR caused light damage and severe apoptosis in liver cells. With the increase of dose, the damage of hepatocyte increased gradually, but the apoptosis of hepatocyte did not increase with the increase of dose of.3.125,6.250,12.500,25.000 g/ (kg. D) microcystin -LR in liver tissue cell apoptosis The index (46.51 + 4.69,11.08 + 3.18,10.72 + 3.36,10.44 + 3.48 respectively) was higher than that of the negative control group (3 + 0.60) (P 0.05). With the increase of -LR dose of microcystin, the expression of Caspase-3 and apoptosis inducible factor mRNA in the liver cells decreased, and the Caspase-3mRNA expression of 3.125,6.250,12.500,25.000 u g / (kg d) was 0.8214 + 0.1150, respectively. The 0.5594 + 0.1800,0.3842 + 0.0745,0.3158 + 0.1307) were lower than those in the negative control group (1) (P 0.05). The dose of microcystin -LR did not affect the expression of G mRNA of hepatocyte endonuclease.
Conclusion: the tumor promoting effect of microcystin -LR may be related to the abnormal expression of Caspase-3 and apoptosis inducing factor mRNA.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R114

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