MC-LTH1降解微囊藻毒素的机理及降解产物的肝细胞毒性研究

Microcystins pollution threatens water ecosystem and public health in China. It's desirable to find effective and safe methods to remove microcystins. A novel indigenous microcystin-degrading bacterium Bordetella sp. MC-LTH1 was isolated and reported for the first time by us (The nucleotide sequence of 16S rDNA was deposited in the Genbank with accession number KC734882). According to the knowledge of Environmental Health, Molecular Biology, Toxicity, and Environmental Chemistry, the microcystin-degrading characteristics of Bordetella sp. MC-LTH1 against three microcystin analogues (microcystin-LR, -RR and -YR) will be studied. The microcystin-degrading activities and molecular-biological mechanisms of this bacterial strain against microcystins will be investigated, through detecting the microcystin-degrading genes, reconstructing genetically engineered bacterium, and expressing the microcystin-degrading substances. The degradation pathway will be analyzed by identifying the degradation products using gas chromatography/high performance liquid chromatography coupled with mass spectrometry (GC/HPLC-MS). Moreover, the individual and joint toxicities of degradation products and microcystins on the human liver cell will be investigated. This study will find new degradation products and mechanisms, Additionally, the study can provide a theoretical basis and technical supports for regulating microcystins pollution by constructing biological method, and assessing environmental safety of this method. Finally, this study is meaningful for achieving effectively and safe removal of microcystins.

微囊藻毒素污染严重威胁着我国水生生态系统和公共卫生安全，如何安全有效去除微囊藻毒素是我们亟待解决的环境科学难题。我们在分离获得一株新型的太湖土著藻毒素降解菌Bordetella sp. MC-LTH1的基础上，拟融合环境卫生学、分子生物学、毒理学、环境化学等多学科知识，研究菌株MC-LTH1对三种微囊藻毒素异构体的降解效果；利用分子生物学技术克隆降解作用相关的基因、构建高效工程菌株和表达降解活性物质，探讨此菌降解微囊藻毒素的分子生物学机制；利用气/液相色谱-质谱连用等技术分离鉴定藻毒素被降解后的产物，分析微囊藻毒素的微生物降解途径；研究微囊藻毒素降解产物的肝细胞毒性，以及降解产物和微囊藻毒素母体混合溶液对肝细胞的联合毒性。本课题的完成可发现新的藻毒素降解产物和分子学机制，为构建生物防治藻毒素污染技术和评价该技术的环境安全性提供科学依据，对实现安全高效去除藻毒素具有重要的理论与实际意义。

目的：探讨细菌对微囊藻毒素的降解解毒机制，并探讨微囊藻毒素及其降解产物的肝细胞毒性及其毒性机制，为构建高效安全的生物防治微囊藻毒素技术提供科学依据与技术支持。材料与方法：分离筛选微囊藻毒素降解菌，在不同pH、温度和微囊藻毒素初始浓度下分析降解菌对微囊藻毒素的降解速率。利用PCR技术扩增降解菌中微囊藻毒素降解基因。通过液相-质谱连用技术测定微囊藻毒素浓度和鉴定微囊藻毒素降解产物。用微囊藻毒素染毒人正常肝细胞HL7702，检测细胞内蛋白磷酸酶2A活性。通过Illumina HiSeqTM2500高通量测序技术检测microRNA的表达量，筛选和预测差异表达的miRNA及其靶基因，并对其靶基因进行GO功能和KEGG通路分析。结果：从太湖中分离出微囊藻毒素降解菌，其降解MC-LR最大速率均可达0.5μg/ml/hr，降解MC-RR速率分别为0.1μg/ml/hr左右。微囊藻毒素降解菌能将MC-LR降解为四肽、Adda和2-甲基-3-甲氧基-4-苯基丁酸MMPB。微囊藻毒素可抑制HL7702细胞内PP2A活性，诱导DNA损伤及微丝重排；影响miRNA的表达，这些差异表达的miRNA，尤其miR-15b-3p，miR-4521和miR-451a在微囊藻毒素导致肝脏损伤的过程中可能发挥了重要作用；miR-451a过表达可一定程度上降低微囊藻毒素诱导的DNA损伤，miR-451a 可能在一定程度上通过对 AKT1表达的调控减小微囊藻毒素诱导的DNA损伤，尚不能确定miR-451a对微囊藻毒素诱导的骨架毒性有保护作用。MC-LR母体对胞内PP2A活性抑制率为82.1 ± 1.3%，当微囊藻毒素被降解菌代谢降解6 h和10 h，微囊藻毒素被降解了80%和100%，其对PP2A活性抑制率为14.9 ± 2.1%和3.9±0.3%。结论：分离筛选的微囊藻毒素降解菌可以高效降解微囊藻毒素，且温度、pH和微囊藻毒素初始浓度等条件可显著影响微囊藻毒素降解速率；获得一种新的藻毒素降解产物MMPB和降解途径；降解菌高效降解微囊藻毒素后，可使微囊藻毒素肝毒性降低，有一定程度的解毒作用。在微囊藻毒素导致肝脏损伤的过程中，一些miRNA，尤其miR-15b-3p，miR-4521和miR-451a可能发挥了重要作用。