微囊藻毒素-LR暴露标志物及凋亡效应的研究

发布时间：2018-07-07 13:16

本文选题：微囊藻毒素-LR + 动物实验　；参考：《华中科技大学》2013年博士论文

【摘要】：第一部分微囊藻毒素-LR暴露生物标志物的研究目的：通过检测动物血液和组织中微囊藻毒素-LR单体及复合物，结合筛查血液细胞可能发生的形态、功能和分子生物学的变化及血液生化指标改变，初步确立能够反映机体暴露藻类毒素污染的生物负荷和生物效应指标，为环境藻类毒素污染与健康评价提供科学实验方法和手段。方法：SPF级雄性昆明小鼠，体重在20~25g之间，用随机数字表法分为5组，每组7只。对照组、低剂量组、中低剂量组、中高剂量组、高剂量组，分别以生理盐水、3.125、6.250、12.500、25.000μg/(kg·d)剂量微囊藻毒素-LR给予腹腔注射染毒。染毒7天后，进行血液生化和血液细胞分析，检测血清细胞因子， KCl-SDS沉淀法检测淋巴细胞DNA-蛋白质交联，流式细胞仪检测单核细胞吞噬功能及单核细胞和淋巴细胞的活性氧，，高效液相色谱-质谱联用技术检测血液及组织中的微囊藻毒素-LR，并分析其变化情况。结果：本研究中各项指标对于微囊藻毒素-LR暴露的敏感度从高至低依次为：肝脏中检测到的微囊藻毒素-LR，巨噬细胞吞噬功能和白细胞活性氧；白细胞介素-6，碱性磷酸酶和乳酸脱氢酶；丙氨酸氨基转移酶和血液中检测到的微囊藻毒素-LR；天门冬氨酸氨基转移酶和肿瘤坏死因子-α。3.125、6.250、12.500、25.000μg/(kg·d)染毒组肝脏中均检测到微囊藻毒素-LR（分别为0.1529±0.0296，0.3047±0.0648，0.6006±0.1105，0.9899±0.2261μg/g肝脏）。同样剂量染毒组淋巴细胞DCF荧光强度（依次为3299.37±120.54，3281.38±58.34，3308.06±136.12，3346.92±108.69）均低于对照组（3770.81±131.39）（P 0.05）；单核细胞DCF荧光强度（依次为3271.51±140.79，3270.05±117.92，3326.90±114.39，3292.49±145.97）均低于对照组（3841.72±130.92）（P 0.05）。6.250、12.500、25.000μg/(kg·d)染毒组小鼠白细胞介素-6（分别为346.837±25.536，360.847±37.886，434.245±35.858pg/mL）均高于对照组（232.775±32.816pg/mL）（P 0.05）；而只有最高剂量25.000μg/(kg·d)染毒组肿瘤坏死因子-α（10.782±0.966fmol/mL）低于对照组（16.878±3.378fmol/mL）（P 0.05）。血清碱性磷酸酶和乳酸脱氢酶在6.250μg/(kg·d)即高于阴性对照组，差异有统计学意义（P 0.05）。结论：本研究对今后的流行病学调查有一定的参考价值，血液和肝脏中检测到微囊藻毒素-LR是暴露的确证标志物，结合血液生化分析、活性氧、细胞因子等的改变可综合分析机体的暴露情况，为微囊藻毒素的研究提供更详细全面的资料。第二部分微囊藻毒素-LR对肝细胞凋亡及Caspase-3、凋亡诱导因子、核酸内切酶G mRNA表达的影响目的：通过观察微囊藻毒素-LR对肝细胞凋亡及Caspase-3、凋亡诱导因子、核酸内切酶G mRNA表达的影响，探讨微囊藻毒素-LR对肝细胞凋亡的影响，丰富微囊藻毒素-LR的凋亡诱导机制。方法：SPF级雄性昆明小鼠，体重在20~25g之间，用随机数字表法分为5组，每组7只。对照组、低剂量组、中低剂量组、中高剂量组、高剂量组，分别以生理盐水、3.125、6.250、12.500、25.000μg/(kg·d)剂量微囊藻毒素-LR给予腹腔注射染毒。染毒7天后，HE染色观察肝脏病理变化，TUNEL染色观察肝细胞凋亡情况，荧光定量PCR检测Caspase-3、凋亡诱导因子、核酸内切酶G mRNA表达变化。结果：小剂量微囊藻毒素-LR暴露导致肝脏细胞出现较轻的损伤和严重的凋亡；随着染毒剂量增加，肝细胞损伤逐渐加重，但肝细胞凋亡并未随染毒剂量升高而增加。3.125、6.250、12.500、25.000μg/(kg·d)微囊藻毒素-LR染毒组肝脏组织细胞凋亡指数（分别为46.51±4.69，11.08±3.18，10.72±3.36，10.44±3.48）均高于阴性对照组（3.00±0.60）（P 0.05）。随着微囊藻毒素-LR染毒剂量的增加，肝细胞Caspase-3、凋亡诱导因子mRNA表达下降；3.125、6.250、12.500、25.000μg/(kg·d)染毒组Caspase-3mRNA表达（分别为0.8214±0.1150，0.5594±0.1800，0.3842±0.0745，0.3158±0.1307）均低于阴性对照组（1.000）（P 0.05）。本研究微囊藻毒素-LR染毒剂量不影响肝细胞核酸内切酶G mRNA表达。结论：微囊藻毒素-LR的促肿瘤作用可能与Caspase-3、凋亡诱导因子mRNA表达异常有关。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

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