【作者】 梁婧；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 微囊藻毒素(microcystin, MC)是一种富营养化水体毒素,化学性质稳定,很难将其从水中除去,而且该毒素具有多种毒性效应,深入研究其毒性机制有着非常重要的意义。MC是一种单环七肽,其中5个氨基酸为固定成分,X,Y为两种可变氨基酸,由此可产生80多种同类物。其中存在较多、毒性较大的是MCLR, L、R分别代表亮氨酸、精氨酸。MCLR是蛋白磷酸酶2A (Protein phosphatase 2A,PP2A)的强效抑制剂,对PP2A的特异性抑制可能是造成其毒作用机制错综复杂的原因之一。然而到目前为止,关于MCLR与PP2A蛋白表达情况及活性的关联研究尚不多见。本实验室前期研究发现MCLR可引起人羊膜细胞FL中PP2A-A的表达升高。为进一步探索PP2A与MCLR毒性效应的相关性,本研究对MCLR暴露后PP2A的改变情况进行了深入探讨。此外,微囊藻毒素的毒性效应有明显的器官选择性,主要表现为肝毒性。这是由于肝脏中表达特殊的有机阴离子转运多肽系统OATPs,可将MCLR大量地转运入细胞。而且近来的研究发现微囊藻毒素对其它不表达或低表达OATPs的脏器也有影响。因此,本研究同时探讨了加入外源性多肽转运体Endo-Porter后MCLR对FL细胞的毒性效应,为今后在不／低表达OATPs细胞中研究MCLR的毒性机制奠定了基础。本研究主要内容包括：MCLR进入细胞后与PP2Ac的结合；MCLR作用后PP2A活性改变及亚基的变化；从微管翻译后修饰的变化研究MCLR染毒后对PP2A调控微管的影响；加入转运体后看MCLR产生的多种毒性效应的变化。采用MTT法检测细胞活力；用实时定量PCR检测mRNA表达的改变情况；用免疫印迹法检测目的蛋白表达的变化；用PI单染法分析细胞周期的改变情况；用Annexin V-PI双染法检测毒素诱导的细胞凋亡率；用免疫荧光技术检测细胞微管形态的变化及目的蛋白相互结合的情况。主要结果：1.短时间低浓度MCLR暴露引起PP2A活性上升,C亚基mRNA和蛋白表达上升,磷酸化水平下降,甲基化水平下降。2. MCLR暴露后微管蛋白表达及形态没有发生变化,但与酪氨酸化微管结合的PP2A-B55a减少。3. MCLR染毒虽然使PP2A多种调节亚基rnRNA发生变化,但并没有导致蛋白表达发生变化。4. MCLR被EP大量转运入细胞后产生一系列毒性效应,抑制PP2A活性、抑制细胞增殖、诱发细胞凋亡、引起ROS水平上升、Bax/Bcl-2比值升高。5. MCLR被EP转运入细胞后,激活ERK1/2/ JNK/ p38。实验进一步证明MCLR激活了MEK1/2-ERK1/2-Myc通路,且呈时间依赖性。6. MCLR被EP转运入细胞后可引起细胞周期阻滞,并同时检测到cdc25C磷酸化水平上升。主要结论：1. MCLR染毒诱发的细胞内毒物兴奋效应使得C亚基mRNA表达和蛋白表达水平上升,从而使PP2A酶活性上升。2. MCLR染毒使PP2A-B55α与酪氨酸化微管的结合能力下降从而影响了PP2A对微管酪氨酸化的调控。3.PP2A的nRNA表达比蛋白表达更容易被毒素影响发生变化。4.通过MCLR被EP大量转运入细胞后产生的一系列毒性效应证明EP可作为MCLR的转运体。5. MCLR被EP大量转运入细胞后激活MEK1/2-ERK1/2-Myc,证明ERK1/2通路在MCLR诱导FL细胞凋亡的过程中起着重要作用。6. MCLR被EP转运入细胞后引起的cdc25C磷酸化水平上升与周期阻滞有关。更多还原

【Abstract】 Microcystins (MCs) are toxins produced by freshwater cyanobacteria that belong to the genera Micocystis, Anabaena, Nodularia, Oscillatoria, and Nostoc. MCs are too stable to degrade naturally and it is difficult to remove them from water. MCs are of increasing importance due to their toxicity and potent tumor promoting activity, so the exact mechanisms of MCs-induced toxicity need further investigation. MCs have been recognized as a family of monocyclic heptapeptide hepatotoxins. Two of the amino acids are variable. Till now, over 80 structural variants have been recognized. Microcystin-LR (MCLR) is the most frequently studied as well as the most toxic in the group of MCs, L and R meaning leucine (L) and arginine(R) respectively. PP2A is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function, such as metabolism, proliferation, differentiation and apoptosis. PP2A holoenzymes are heterotrimers consisting of a core dimmer, AC, and a regulatory B-type subunit. MCLR has been recognized as the strong inhibitor of PP2A, which may be one reason of the implicated intoxication mechanism of MCLR. Studies in our laboratory have revealed that exposure to MCLR, MCRR and bloom extract could increase the protein levels of the A subunit of PP2A. Therefore, the present study was designed to illustrate the precise events happening in MCLR-induced toxicity in particular concerning with PP2A. Furthermore, the liver is the prime target organ affected by MCLR. A group of hepatocyte uptake transporters termed organic anion transporting polypeptides (OATPs) present mainly in liver are crucial for MCLR transferring into cells. In addition, it should be of great concern that accumulation of MCs in other organs without expression of OATPs has also been reported in recent studies. Therefore, to investigate the mechanism of MCLR toxicity in other cells without/low OATPs expression, we testified an alternative approach to study the toxicity of MCLR using the facilitated transporter Endo-Porter (EP)-a peptide reagent binding to cell membrane.The present study was focused on the binding of MCLR to PP2Ac, the effects of MCLR on PP2A activity and subunits, the regulation of PP2A on the post-translational modification of microtubule after MCLR treatment and the cytotoxicity of MCLR in the presence of Endo-porter in FL cells. MTT was used to evaluate cell viability; real-time PCR and western blot were used to detect the expression level of mRNA and protein respectively; cell cycle and apoptosis were measured by PI staining and Annexin V-PI staining; immunofluorescence was used to detect the co-locolization of aimed proteins and morphology of microtubule.The results were shown as below:1. MCLR treatment increased the activity of PP2A as well as the expression levels of mRNA and protein of PP2Ac, decreased the phosphorylation and methylation of PP2Ac.2. Exposure to MCLR had no effect on FL cells morphology and protein level of a tubulin. However, the binding of B55a and tryosinated tubulin was significantly reduced after MCLR treatment.3. MCLR exposure changed the levels of mRNA of PP2A regulatory subunits, however, exposure to MCLR had no significant effects on the protein levels.4. The accumulation of MCLR in FL cells in the presence of EP induced toxic effects including decrease of PP2A activity, inhibition of cell viability, increase of apoptotic rate, and increased generation of intracellular ROS and the elevation of Bax/Bcl-2 ratio.5. The present study revealed that MCLR could activate ERK1/2, JNK and p38. Furthermore, MCLR activated MEK1/2-ERK1/2-Myc pathway in a time-dependent manner. 6. MCLR exposure in the presence of EP induced cell cycle arrest and up-regulation of phosphrylated cdc25C at Ser216.The conclusions were shown as below:1. It is supposed that the hormesis response induced by MCLR increased the expression levels of mRNA and protein of PP2Ac, resulting in the increased activity of PP2A.2. The changed levels of mRNA of PP2A regulatory subunits by MCLR treatment suggest that the expression level of mRNA is more sensitive to MCLR treatment than that of protein.3. Exposure to MCLR influences the regulatory role of PP2A-B55αon tryosinated tubulin.4. The toxic effects of MCLR in FL cells in the presence of EP suggest that the utility of EP might be of great help for elucidating the complicated toxicity of MCLR in vitro.5. The ERK1/2 pathway might play an important role in the apoptosis induced by MCLR in the presence of EP.6. The up-regulation of phosphrylated cdc25C at Ser216 could contribute to the cell cycle arrest induced by MCLR in the presence of EP.更多还原