新型荧光探针法研究食药用真菌多糖生物合成与细胞内pH的关系

Polysaccharides isolated from edible and medicinal fungi have exhibited many important bioactivities such as antitumor, immune modulatory, anticoagulation, lowering blood lipids and anti-aging effects. However, the industrialization of polysaccharide biosynthesis by edible and medicinal fungi is subject to low yield because their biosynthesis mechanism is still unclear. Thus clear mechanism of polysaccharide biosynthesis is very important to help effectively improving yields of polysaccharides. It is supposed that polysaccharide biosynthesis should be closely related to intracellular pH because intracellular pH is an important factor to regulate all the biological reactions in living cells. Therefore, the aim of the current project is firstly to develop a new method of intracellular pH measurements for edible and medicinal fungi cells with high selectivity and sensitivity, and the key of the method is to synthesize new pH ratio fluorescent probes based on carbon dots. Secondly, polysaccharide biosynthesis will be regulated by means of extracellular condition alterations, and then the change of intracellular pH along with the regulation will be dynamically measured by the method of intracellular pH measurement being developed in this project. Consequently, the relationship between the biosynthesis of polysaccharides and intracellular pH will be finally elucidated. It is of scientific interest that the current project will provide theoretical and experimental basis for understanding the mechanism of polysaccharide biosynthesis in fungi cells. Wonderful results will be achieved when basic scientific research on edible and medicinal fungi is performed creatively using the new ideas and methods of material science and chemistry.

食药用真菌多糖具有抗肿瘤、抗病毒、免疫调节、抗凝血、降血脂、延缓衰老等重要的生物活性。然而，低产率限制了食药用真菌多糖生物合成的工业化。显著提高产率的必要前提是阐明影响食药用真菌多糖生物合成的内在机理，而研究多糖生物合成与细胞内pH的关系是阐明影响食药用真菌多糖生物合成内在机理的关键。由此可见，本项目研究不仅科学问题明确，而且具有重要的理论意义。本项目研究内容富有创新，特色鲜明：设计、构建基于碳量子点的新型pH比率荧光探针，利用该类探针建立准确、动态、无损伤地测定食药用真菌细胞内pH的新方法；通过胞外环境宏观调控食药用真菌多糖的生物合成，利用所建立的测定细胞内pH新方法对调控过程中真菌细胞内pH进行动态测定，考察食药用真菌多糖生物合成与细胞内pH的关系。本项目创新性地利用材料学、化学的新观点和新方法研究食药用真菌中的基础科学问题，有望取得重要的研究成果。

食药用真菌多糖具有多种重要的生物活性，日益受到人们的高度重视。阐明多糖生物合成内在机理是提高食药用真菌多糖产率的必要科学前提，而真菌多糖生物合成与细胞内pH的关系是影响真菌多糖生物合成的关键因素。因此，本项目首先合成了基于碳量子点新型pH荧光探针，考察其对食药用真菌的毒性，优化得到探针对菌丝的成像条件，建立了真菌胞内pH的测定方法，调节真菌胞内多糖含量并测其胞内pH，建立真菌多糖与胞内pH值的关系。.（1）以苏氨酸，间苯二胺和PEG1500为原料分别合成了发射蓝色（T-CDs）和绿色（G-CDs）荧光的碳量子点，以此构建了两种单峰pH荧光探针。利用丙烯酸和乙二胺合成蓝光碳量子点并共价键合异硫氰酸荧光素，构建基于碳量子点的比率荧光探针（CD-FITCs）。以柠檬酸、聚乙烯亚胺和罗丹明B为原料一步热解法合成pH比率荧光探针(CA-RhB-PEI-CDs)。利用聚乙二醇400作为交联剂，将柠檬酸、1, 4-丁二胺合成蓝光发射碳量子点(CA-BDA-CDs)与罗丹明B和苯胺蓝连接形成比率pH荧光探针(ANI-CA-BDA-CDs)。所合成荧光探针荧光强度或比率值均在生理pH条件下与pH值呈现良好的线性关系。.（2）通过生长速率方法研究pH探针G-CDs，CA-RhB-PEI-CDs和ANI-CA-BDA-CDs对黄伞和海鲜菇真菌的细胞毒性，结果表明pH探针对真菌细胞无细胞毒性和良好的生物相容性。优化了G-CDs和ANI-CA-BDA-CDs探针对食用真菌黄伞和海鲜菇的荧光成像条件。利用尼日菌素的高K+ HEPES缓冲溶液中进行细胞内校准实验，使用Image J软件处理荧光图像，建立细胞内pH的校准曲线。.（3）利用光照和黑暗条件，以及G-CDs和以柠檬酸和乙二胺为原料合成的蓝光碳量子点对黄伞和海鲜菇胞内多糖的生物合成进行调节。碳量子点在合适的浓度对胞内多糖的生物合成具有促进作用。黑暗条件相对于光照条件下更有利于胞内多糖的生物合成。利用上述确立的比率荧光探针法动态测定调控过程中食药用真菌细胞内pH，建立两者之间的关系。