P-糖蛋白在栉孔扇贝对麻痹性贝类毒素累积和排出过程中的作用研究

Many aquatic species are able to survive in environments which contain high levels of multiple anthropogenic pollutants or natural product toxins. This multixenobiotic resistance (MXR) phenomenon, existed in many kinds of aquatic organisms, is similar to multidrug resistance (MDR) first observed in tumor cell lines resistant to anti-cancer drugs. MXR proteins, like P-glycoprotein (P-gp), may actively export natural product toxins and anthropogenic pollutants out of cells or facilitate the sequestration of toxins within specialized cells or organelles, effectively compartmentalizing them away from target molecules, and enhance the resistance or tolerance aquatic organisms to multixenobiotic substances. Studies on the function(s) of MXR in the resistance or tolerance to the paralytic shellfish poisoning toxins (PSP toxins) in bivalves are rare..This project is planning to carry out researches on the transporter activity dynamic changes of P-gp in the process of PSP toxin accumulation and depuration after exposing the scallop Chlamys farreri to PSP toxin-producing marine algae Alexandrium tamarense. The correlation and concentration-response relationship between P-gp transporter activity and PSP toxin accumulation and depuration will be clarified. On this basis, by the immunohistochemical analysis, the distribution of P-gpin the scallop tissue will be determined. Whether PSP toxins bind with P-gp and whether transmembrane transportation happens will also be studied by isolating the membrane and hydrolyzing it by suitable protease. All these above studies will attributed to expound whether P-gp plays a role in the toxin accumulation and depuration. This project finally aims to reveal the possible role of P-gp for PSP resistance or tolerability in shellfish toxin. These researches will rich the PSP toxins tolerance mechanism research in shellfish, and it will provide the scientific basis and foundation for PSP toxins management, disaster prevention and mitigation.

多型异源物质抗性机制是贝类等水生生物中广泛存在的一种特殊跨膜转运机制，类似于哺乳动物肿瘤细胞中的多药抗药机制。跨膜转运蛋白如P-糖蛋白（P-gp）通过将异源物质运出细胞或促进异源物质在特定的细胞或细胞器内滞留或隔离，防止异源物质与细胞内的靶分子结合来提高生物体的抗性和耐受性。关于多型异源物质抗性机制在贝类耐受麻痹性贝毒方面的研究鲜见报道。本项目拟通过毒素暴露实验，研究在毒素累积和排出过程中栉孔扇贝体内P-gp转运活性的动态变化，阐明PSP毒素累积和排出与P-gp活性的相关性和量效关系；在此基础上，对P-gp进行免疫组织定位，确定P-gp高表达组织，分离该组织细胞膜，研究P-gp与PSP毒素分子是否结合，明确P-gp在转运PSP毒素中作用。本项目旨在揭示P-gp在贝类对PSP毒素的抗性或耐受性中的可能作用，丰富贝类PSP毒素耐受机制研究，并为PSP毒素管理、防灾减灾提供科学依据和基础。

多型异源物质抗性机制是贝类等水生生物中广泛存在的一种特殊跨膜转运机制，类似于哺乳动物肿瘤细胞中的多药抗药机制。跨膜转运蛋白如P-糖蛋白（P-gp）通过将异源物质运出细胞或促进异源物质在特定的细胞或细胞器内滞留或隔离，防止异源物质与细胞内的靶分子结合来提高生物体的抗性和耐受性。关于多型异源物质抗性机制在贝类耐受食源性毒素方面的研究鲜见报道。本项目以栉孔扇贝为受试生物，通过麻痹性贝类毒素（PSP毒素）和虾夷扇贝毒素（YTX毒素）暴露实验，研究了毒素累积和转化过程中栉孔扇贝体内P-gp转运活性的动态变化，阐明了毒素累积和转化与P-gp活性的相关性；在此基础上，采用qRT-PCR方法和免疫组织定位，分别在分子和水平和蛋白水平研究了毒素暴露对P-gp及其他三种与解毒和抗性相关的蛋白（多药耐药相关蛋白-MRP1，谷胱甘肽巯基转移酶-GST1，细胞色素P450 3A蛋白- CYP3A)基因mRNA表达的影响及蛋白在扇贝组织中的分布情况，初步探讨了P-gp在栉孔扇贝耐受PSP毒素和YTX毒素中可能的作用。研究结果表明，P-gp在贝类耐受PSP毒素和YTX毒素方面发挥了一定的作用，但这种作用具有一定的种属差异和组织差异。项目基本完成了预期设定的目标。项目的完成为揭示P-gp在贝类对食源性毒素的抗性或耐受性中的可能作用，丰富贝类耐受机制研究及海洋生物毒素管理、防灾减灾方面都具有重要的理论意义和实际意义。