微囊藻毒素引起睾丸支持细胞紧密连接损伤的分子机制及其干预的基础研究

The tight junction of testicular Sertoli cell plays an important role in the development and maturation of sperm. In our previous studies, we have found that Microcystins (MCs) which produced by freshwater cyanobacteria can be transported into Sertoli cell, causing injury to the tight junction. As we know, the function of tight junction is regulated by PI3K/AKT signaling pathway. We speculated that MC induced tight junction injury through this pathway. On the basis of preliminary work, the project is proposed to carry out the following research: ①Establishing the Sertoli cell Oatp super family expression profiling `and confirming the exactly carrier protein which mediated MC-LR into Sertoli cell by CRISPR/Cas9 technology; ② Establishing the MC-LR exposed Sertoli cell model, ascertain the molecular mechanism of MCs-induced tight junction injury based on PI3K/AKT signaling pathway; ③Establishing the MC-LR exposed male mouse mode, to illustrate the molecular mechanism of MC-LR cause tight junction damage; ④Use specific targeting inhibitors of PI3K/AKT signaling pathway, to protect tight junction integrity from MC-LR. Ascertain its possible regulatory mechanism in tight junctions of MCs injured testicular cells, which might facilitate us to use specific method for intervening MCs induced tight junction toxic, bringing about great significance for male reproductive health protection.

睾丸支持细胞紧密连接对精子的正常发育成熟具有至关重要的作用。申请人在前期研究中发现淡水蓝藻产生的天然毒素-微囊藻毒素（MCs）能够进入支持细胞，损伤支持细胞紧密连接。已知紧密连接受PI3K/AKT信号通路调控，我们猜测MCs可能影响该通路进而破坏支持细胞紧密连接。本项目拟在前期工作基础上，开展以下研究：①建立支持细胞Oatp超家族成员表达谱，采用CRISPR/Cas9技术，确认介导MC-LR进入支持细胞的载体蛋白；②建立MC-LR染毒支持细胞模型，基于PI3K/AKT信号通路探索MC-LR引起支持细胞紧密连接损伤的分子机制；③建立MC-LR染毒雄性大鼠模型，在体研究MC-LR引起支持细胞紧密连接损伤分子机制；④利用PI3K/AKT信号通路特异性抑制剂，研究其对MC-LR引起紧密连接损伤的保护作用。研究结果对于进一步揭示MC-LR的生殖毒性分子机制以及治疗药物的开发具有一定的指导意义。

睾丸支持细胞紧密连接对精子的正常发育成熟具有至关重要的作用。本项目通过不同的手段获得如下实验结果 ①建立支持细胞Oatp超家族成员表达谱，采用siRNA干扰技术，确认Oatp3a1介导MC-LR进入支持细胞；②建立MC-LR染毒支持细胞模型，确认MC-LR通过PI3K/AKT信号通路引起支持细胞紧密连接和缝隙连接损伤；③建立MC-LR染毒雄性大鼠模型，在体内进一步确认MC-LR引起支持细胞紧密连接和缝隙连接损伤分子机制是通过PI3K/AKT信号通路；④利用PI3K/AKT信号通路特异性抑制剂和ICA可抑制MC-LR引起紧密连接损伤。研究结果对于进一步揭示MC-LR的生殖毒性分子机制以及治疗药物的开发具有一定的指导意义。