低剂量微囊藻毒素对原代大鼠肝脏细胞的促增殖效应及其初步机制研究

发布时间：2018-05-15 04:13

本文选题：微囊藻毒素 + 细胞凋亡　；参考：《复旦大学》2011年硕士论文

【摘要】：近年来,水华现象在世界各地频繁发生,给人类健康带来严重的威胁。微囊藻毒素(Microcystin, MC)是一类蓝藻水华产生的具有生物毒性的单环七肽化合物,至今己发现80余种异构体。MC性质稳定,难以降解,现行自来水处理工艺不能将其有效去除。此外,MC还具有生物富集作用,可通过食物链进入人体。MC通过饮水或饮食对人体健康构成的危害已经引起世界各国的广泛关注。MC生物毒性作用的靶器官主要为肝脏。有研究表明,MC的毒性效应表现为剂量依赖性的双向毒性,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则促进细胞增殖以及肿瘤的形成。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。目前有关MC的毒性机制研究大多集中于其诱导细胞凋亡的分子机制,而其促癌机制的研究国内外并不多见。动物实验研究结果显示,MC是肿瘤发生的促进剂而非引发剂,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其促癌机制具有重要意义。本课题基于目前MC的研究现状以及存在的问题,选取毒性最强、产生量最大和危害最严重的微囊藻毒素MC-LR,染毒原代大鼠肝脏细胞,从细胞生物学与分子生物学的角度研究MC-LR对原代大鼠肝脏细胞的毒性效应。系统性探讨了MC-LR特异性肝脏毒性的作用特征,在明确MC-LR具有诱导细胞凋亡和促进细胞增殖的双向毒性及其剂量范围的基础上,进一步分析MC-LR促细胞增殖效应的可能机制,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；同时,也为进一步深入探讨MC的促癌分子机制提供研究线索。研究采用胶原酶灌流法建立了原代大鼠肝脏细胞培养模型,通过监测培养液中肝细胞受损特征性酶谱一谷丙转氨酶(ALT)和谷草转氨酶(AST)的水平,以及对肝细胞表面特异性标志物的分析,证实分离得到的肝细胞完整性良好且纯度达到近100%。从而建立了适用于课题研究的实验模型,为后续实验现象的观察和指标的测定奠定了基础。通过染毒10-5～10-12 mol/L梯度浓度的MC-LR,观察其在较长染毒时间范围内(24 h-72 h)的毒性作用特征,发现MC-LR对原代大鼠肝脏细胞存在剂量依赖性的双向毒性作用,即μM级别的MC-LR对原代大鼠肝脏细胞具有细胞毒性作用,表现为细胞状态恶化、细胞活率显著下降、细胞凋亡率明显上升；而pM～nM级别的MC-LR具有促进细胞增殖效应,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。进一步对MC-LR导致的原代大鼠肝脏细胞增殖效应进行研究,发现MAPK家族成员磷酸化状态、细胞内ROS含量以及抗氧化系统均与这一效应有关。具体表现为：MAPK家族的磷酸化状态与低剂量(10-9～10-12 mol/L) MC-LR的促细胞增殖效应密切相关,在细胞增殖活跃的时问(48 h以内),ERK1/2的磷酸化活性显著增加,而JNK1/2和p38的磷酸化活性受到了明显的抑制；10-9～10-12mol/L浓度的MC-LR染毒后,细胞内ROS水平出现了温和的变化,低水平ROS对大鼠肝脏细胞反复而温和的刺激与MC-LR促增殖效应的时间区间(48 h以内)存在很大的重叠性,且与细胞周期、MAPK结果相一致。
[Abstract]:In recent years, the phenomenon of water bloom occurs frequently in all parts of the world and poses a serious threat to human health. Microcystin (MC) is a kind of biologically toxic mono ring seven peptide produced by cyanobacteria bloom. Up to now, more than 80 isomers have been found to be stable and difficult to degrade. The current tap water treatment process can not be effective. In addition, MC also has bioaccumulation, and can enter the human body's health by drinking water or diet through the food chain through the food chain. The target organs of the world's widespread concern about the biological toxicity of.MC are mainly liver. Studies have shown that the toxic effects of MC are dose dependent bi-directional toxicity, that is, high dose of MC. The amount of MC can cause programmed cell death or apoptosis, while low doses of MC promote cell proliferation and tumor formation. Because the harm of MC to the population is more concentrated on long-term low dose exposure through drinking water or diet, and more and more epidemiological data show its correlation with the occurrence of swollen tumors, so the chronic toxicity of MC is especially significant. The potential carcinogenicity has become one of the hotspots of research. At present, the research on the toxicity mechanism of MC is mostly focused on the molecular mechanism of inducing cell apoptosis, and the research on the mechanism of cancer promoting is not common at home and abroad. The results of animal experiments show that MC is a promoter rather than a initiator, and the proliferation of out of control cells is in the swelling. Therefore, studying the effect of MC on cell proliferation is of great significance in elucidating its mechanism of promoting cancer.
Based on the current research status of MC and the existing problems, we select the most toxic, the largest and most serious microcystin MC-LR, dye the primary rat liver cells, and study the toxic effect of MC-LR on the primary rat liver cells from the angle of cell biology and molecular biology. The MC-LR specificity is systematically discussed. On the basis of identifying the bidirectional toxicity and dose range of MC-LR inducing cell apoptosis and promoting cell proliferation, the possible mechanism of MC-LR promoting cell proliferation effect is further analyzed to provide experimental basis and theoretical basis for clarifying the relationship between MC and high incidence of liver cancer. MC provides a clue to the molecular mechanism of cancer promoting.
The primary rat liver cell culture model was established by collagenase perfusion method. By monitoring the level of ALT and AST, and the analysis of the specific markers on the surface of the liver cells, the integrity and purity of the liver cells were obtained. Near 100%., an experimental model suitable for research was established, which laid a foundation for subsequent observation and index determination.
The toxicity of 10-5 to 10-12 mol/L gradient concentration was observed. The toxic effects of MC-LR on the primary rat liver cells were observed in a long period of time (24 h-72 h), and it was found that MC-LR had a dose-dependent two-way toxic effect on the primary rat liver cells. That is, the MC-LR of the M grade MC-LR has cytotoxic effect on the primary rat liver cells, which shows the cell status. The rate of cell viability decreased significantly and the rate of cell apoptosis increased obviously, while the MC-LR of pM ~ nM level had the effect of promoting cell proliferation, the increase of cell viability, the increase in the proportion of S cells in the cell cycle and the increase of DNA synthesis rate.
Further studies on the proliferation of primary rat liver cells induced by MC-LR show that the state of phosphorylation of MAPK family members, the content of ROS in the cells and the antioxidant system are all related to this effect. The specific expression is that the phosphorylation status of the MAPK family is closely related to the proliferation effect of low dose (10-9 to 10-12 mol/L) MC-LR. When the cell proliferation was active (within 48 h), the phosphorylation activity of ERK1/2 increased significantly, while the phosphorylation activity of JNK1/2 and p38 was significantly inhibited, and the intracellular ROS level appeared mild changes after 10-9 ~ 10-12mol/L concentration of MC-LR, and low level ROS had repeated and mild stimulation with MC-LR to promote proliferation of rat liver cells. The time interval of the effect (within 48 h) is very overlapped, and is consistent with the cell cycle and MAPK results.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【参考文献】