低剂量微囊藻毒素对原代大鼠肝细胞促增殖效应的分子机制研究

发布时间：2018-03-23 16:27

本文选题：微囊藻毒素　切入点：原代大鼠肝细胞　出处：《复旦大学》2013年硕士论文

【摘要】：随着水华污染的全球流行,人群暴露藻毒素的机会大大增高,对藻毒素的研究也受到越来越多的关注。微囊藻毒素(microcystin, MC)是由水体中的蓝藻产生的具有生物活性的单环7肽化合物,目前已发现了90余种MC的异构体。大量研究显示长期饮用低浓度MC污染的水与原发性肝癌、大肠癌和胃肠道肿瘤等肿瘤的高发率有关。MC生物毒性作用的靶器官为肝脏。已有的实验研究提示,MC的毒性效应表现为剂量上的双效性,即高剂量的MC能引起细胞程序性死亡或凋亡,而低剂量的MC则具有促进细胞增殖以及肿瘤形成的效应。由于MC对人群的危害更多地集中于通过饮水或饮食长期低剂量的暴露,且越来越多的流行病学资料显示其与肿瘤发生的相关性,因此,MC的慢性毒性尤其是潜在的致癌性成为研究的热点之一。多数动物实验研究显示,MC是肿瘤发生的促长剂而非引发剂,而失控的细胞增殖是在肿瘤形成过程中至关重要。因此,研究MC的促细胞增殖效应对于阐明MC的促癌作用机制具有重要意义。课题组前期研究发现1×10-5～1×10-6mol/L的MC-LR可引起细胞凋亡的发生；而1×10-9～1×10-12mol/L的MC-LR则具有促细胞增殖的现象,表现为细胞活率增加、细胞周期中S期细胞比例增加,DNA合成速率增加。基于此,本课题继续采用原代大鼠肝脏细胞为研究模型,深入对低剂量MC-LR的促细胞增殖效应进行研究,在更早更多的时间点观察MC促细胞增殖效应及可能的分子机制,从细胞生物学与分子生物学的角度探讨MC低剂量暴露下原代大鼠肝细胞细胞周期及周期调控蛋白的变化,分析与细胞增殖密切相关的MAPK信号通路改变,为阐明MC与肝癌高发之间的关系提供实验基础和理论依据。研究发现以1×10-9～1×10-12mol/L的MC-LR染毒原代大鼠肝细胞可引起细胞增殖效应,细胞增殖效应在染毒12h即能被观察到且在1×10-11mol/L组增殖效应明显,表现为细胞活率增加、细胞周期中S期细胞比例增加。进一步研究发现,细胞周期相关蛋白cyclin D1、cyclin E mRNA及蛋白水平在MC-LR染毒后(3h-6h)可观察到有增高表现,cyclin A在染毒后(6h-12h) mRNA及蛋白水平增高。MAPK家族的P44/42、JNKl/2、P38的磷酸化活性在MC-LR染毒后(3h-6h)可观察到有增高表现,MAPK下游转录因子AP-1亚基c-fos在mRNA、蛋白及其磷酸化状态三个层面活性均在MC-LR染毒后(3h-6h)观察到增高表现。c-fos蛋白及磷酸化状态高水平持续至12h,c-fos mRNA水平在12h呈下降趋势,24h c-fos各指标回复对照组水平。c-jun三个层面高表达的效应持续存在于染毒后3h-24h。各分子效应最明显在1×10-10或1×10-11mol/L剂量组,与细胞增殖效应在剂量组上重合,早于细胞增殖现象且在作用时间上相互呼应。实验结果提示细胞周期蛋白cyclin的高表达、MAPK家族蛋白磷酸化活性的增高、AP-1活性增强可能与MC-LR的促细胞增殖效应相关。
[Abstract]:With the global epidemic of Shui Hua's pollution, the chance of exposure of the population to algal toxins has greatly increased. Microcystins (MCCs) are biologically active monocyclic 7-peptide compounds produced by cyanobacteria in water. At present, more than 90 kinds of MC isomers have been found. A large number of studies have shown that drinking water contaminated with low concentrations of MC for a long time and primary liver cancer, The high incidence rate of colorectal cancer and gastrointestinal cancer. The target organ of the biological toxicity of MC is liver. That is, high dose MC can induce programmed cell death or apoptosis, while low dose MC has the effect of promoting cell proliferation and tumor formation. And more and more epidemiological data show its correlation with tumorigenesis, Therefore, the chronic toxicity of MC, especially its potential carcinogenicity, has become one of the hot topics. Most animal studies have shown that MMC is a promoter rather than an initiator for tumorigenesis. And uncontrolled cell proliferation is crucial in tumorigenesis. It is important to study the effect of MC on promoting cell proliferation in order to elucidate the mechanism of MC's carcinogenesis. Our previous study found that 1 脳 10 ~ (-5) MC-LR with 1 脳 10 ~ (-5) 10-6mol/L could induce apoptosis, while 1 脳 10 ~ (-9) MC-LR with 1 脳 10 ~ (-9) 10-12mol/L could promote cell proliferation. The results showed that the cell viability was increased and the proportion of S phase cells increased in cell cycle. Based on this, we continue to study the effect of low dose MC-LR on cell proliferation by using primary rat liver cells as the research model. The effects of MC on cell proliferation and its possible molecular mechanism were observed at earlier and more time points. The changes of cell cycle and cycle regulatory proteins in primary rat hepatocytes under low dose exposure of MC were studied from the point of view of cell biology and molecular biology. The changes of MAPK signaling pathway closely related to cell proliferation were analyzed to provide experimental and theoretical basis for elucidating the relationship between MC and the high incidence of hepatocellular carcinoma. It was found that the primary cultured rat hepatocytes exposed to 1 脳 10 ~ (-9) ~ 1 脳 10-12mol/L MC-LR could induce cell proliferation, which could be observed at 12 h after exposure to 1 脳 10 ~ (-9) 10-12mol/L, and could be observed in 1 脳 10 ~ (-9) 10-11mol/L group, which showed that the cell viability was increased. The proportion of S phase cells increased in cell cycle. The levels of cyclin E mRNA and protein of cyclin D1C gene in the cell cycle related protein were increased after MC-LR exposure (3h-6h). The increased level of mRNA and the protein level. The phosphorylation activity of P44 / 42JNKl2 / P38 in the MC-LR family was observed to be increased after MC-LR exposure (3h-6h). The activities of AP-1 subunit c-fos, a transcription factor downstream of high expression MAPK, in mRNAs, proteins and their phosphorylation state were observed at 3h-6 h after MC-LR exposure. The high level of .c-fos protein and phosphorylated state continued until 12h after exposure to MC-LR. The level of c-fos mRNA decreased gradually. The effect of high expression of c-jun on the three levels of c-fos returned to the control level at 24 h after exposure was observed at 3h-24 h after exposure. The molecular effects were most obvious in the 1 脳 10 ~ (-10) or 1 脳 10-11mol/L groups. The effect of cell proliferation coincided with that of cell proliferation in the dose group. The results indicated that the high expression of cyclin cyclin family protein phosphorylation activity and the increase of AP-1 activity may be related to the effect of MC-LR on cell proliferation.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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