【作者】 黄夏宁；

【导师】 谷康定；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2013， 博士

【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。更多还原

【Abstract】 Part1: Biomarkers of Expoure to Microcystin-LR in MiceObjective: In order to investigate the response indices to toxic microcystin-LR in blood ofmice, concentrations of free and total microcystin-LR in blood and tissues, accompanied byserous parameters in series, including some enzymatic activities, hematology and thefunction of leukocytes, were determined.Methods: SPF（specific pathogen free） Kunming male mice, weighting20~25g, wererandomly divided into5groups,7mice for each group. The mice in5groups were exposedto microcystin-LR through intraperitoneal injection at0.000,3.125,6.250,12.500and25.000μg/kg body weight respectively for7days. Then the blood samples wereautomatically analyzed by the instrument for the serum biochemical indices and thehematological investigations; cytokine levels in the serum were measured byradioimmunoassay; the measurement of DNA-protein crosslinks was performed using theSDS/KCl precipitation technique; the phagocytosis and reactive oxygen species ofleukocytes was detected by flow cytometry; and the concentrations of free and totalmicrocystin-LR in blood and tissues were determined by the liquid chromatography-massspectrometry method.Results: The positive and sensitive findings in this experiment could be divided into fourgroups. The most sensitive indices were total microcystin-LR in the liver, phagocytic indexand reactive oxygen species. The toxin in the liver was reliable and powerful, phagocyticindex was affected by some subject factors and reactive oxygen species lacked a dose-effect relationship. The second sensitive indices were interleukin-6, alkaline phosphatase andlactate dehydrogenase, all of which showed good dose-effect results. The third were alanineaminotransferase and free microcystin-LR in the blood, of which the former was relativelyless sensitive and specific, and the latter was specific and reliable but less sensitivecompared with the toxins in the liver. The least sensitive indices were aspartate transaminaseand tumor necrosis factor-α. The total microcystin-LR in the four dose-treated groups were0.1529±0.0296，0.3047±0.0648，0.6006±0.1105and0.9899±0.2261μg/g liver weight,respectively. The reactive oxygen species levels of lymphocytes (3299.37±120.54,3281.38±58.34,3308.06±136.12and3346.92±108.69, respectively) in the4treated groupswere significantly lower than those in the control group (P <0.05); and the reactive oxygenspecies levels of monocytes (3271.51±140.79,3270.05±117.92,3326.90±114.39and3292.49±145.97, respectively) in the4treated groups also decreased significantly comparedwith the control group (P <0.05). The levels of interleukin6(346.837±25.536,360.847±37.886and434.245±35.858pg/mL, respectively) in the6.250,12.500and25.000μg/(kg·d) dose group rose in proportion, higher than those in the control group (P <0.05);while the levels of tumor necrosis factor-alpha (10.782±0.966fmol/mL) in25.000μg/(kg·d)dose group were statistically lower than those in the control group (P <0.05). The levels ofalanine aminotransferase (187.99±53.00and381.54±89.23U/L, respectively) in the12.500and25.000μg/(kg·d) dose group were on the significant rise, higher than those in the controlgroup (P <0.05). The levels of aspartate transaminase (473.10±168.07U/L) in the25.000μg/(kg·d) dose group were significantly higher than those in the control group (P <0.05).Thelevels of alkaline phosphatase (128.54±21.46,170.43±22.11and223.26±66.35U/L,respectively) and lactate dehydrogenase (690.50±112.11U/L，769.00±136.92U/L and952.53±169.73U/L, respectively) in the6.250,12.500and25.000μg/(kg·d) dose groupwere all above the control group, showing statistical differences (P <0.05).Conclusion: The results of the studies suggest that measurement of microcystin-LR in bloodis powerful and clear evidence to indicate that the subjects have been exposed tomicrocystin-LR and can be used to discriminate from other causes leading to hepatic lesionsalthough the microcystin-LR in blood is not as sensitive as other indices that are usually lessspecific but are useful complements to reflect the liver function. Part2: The Role of Microcystin-LR in Inducing Apoptosisof Liver Cells and its Relationship with the mRNAExpressions of Caspase-3, Apoptosis Inducing Factor andEndonuclease GObjective: The main purpose of this study was to investigate the apoptotic effects caused bymicrocystin-LR and its possible influence on the mRNA expressions of caspase-3, apoptosisinducing factor and Endonuclease G in mice liver.Methods: SPF（specific pathogen free） Kunming male mice, weighting20~25g, wererandomly divided into5groups,7mice for each group. The mice in5groups were exposedto microcystin-LR through intraperitoneal injection at0.000,3.125,6.250,12.500and25.000μg/kg body weight respectively for7days. HE staining, TUNEL and real time PCRwere used to detect the histopathological changes, cell apoptosis as well as the mRNAexpression of caspase-3, apoptosis inducing factor and Endonuclease G in mice liver.Results: Compared to the control group, severe histopathological changes and significantdifferences in cell apoptosis were observed in3.125,6.250,12.500and25.000μg/(kg·d)dose groups. And further examinations showed that the mRNA expressions of caspase-3andapoptosis inducing factor were decreased in a dose-response relationship. There was nosignificant difference in the mRNA expressions of Endonuclease G among the5dose groups.The levels of apoptosis index by TUNEL assay(46.51±4.69，11.08±3.18，10.72±3.36and10.44±3.48, respectively) in the3.125,6.250,12.500and25.000μg/(kg·d) dose group weresignificantly higher than those in the control group (P <0.05). And the mRNA expressionsof Caspase-3(0.8214±0.1150，0.5594±0.1800，0.3842±0.0745and0.3158±0.1307,respectively) in the3.125,6.250,12.500and25.000μg/(kg·d) dose group were significantlylower than those in the control group (P <0.05).Conclusion: The present study has demonstrated that microcystin-LR has relevance to theabnormal mRNA expressions of caspase-3and apoptosis inducing factor in the liver.更多还原