CD147-MCTs调控乳酸穿梭在低剂量铅致睾丸细胞毒性中的作用研究

Poor sperm quality caused by environmental lead pollution is an important problem. Its mechanism is not clear. In spermatogenesis, lactate is the central energy metabolite and anti-apoptotic factor. Lactate is produced by somatic Sertoli cells, and then utilized primarily by the germ cells. Analysis of the literature and our preliminary study prompted that the Pb-induced testicular toxicity involve in the interference of intracellular lactate transport. The intracellular lactate shuttle mediated by CD147 cooperation with the monocarboxylate transporter (MCTs), may be the key target. The objective of this study is to investigate the effect of CD147-MCTs mediated lactate shuttle in the improvement of testicular toxicity induced by low-dose lead exposure. We will determine that (1) the dose- and time-dependent effects of Pb treatment on lactate level and the expression levels of CD147, MCTs and CD147-MCTs in the Sertoli cells, spermatocytes and non contact co-culture system Sertoli cells and spermatocytes. (2) the relationship between the change of lactate level, CD147-MCTs expression levels and the toxic effect of lead on spermatocyte.（3）application of the cell lines with different expression levels of CD147 and MCTs obtained by biotechnology, and non contact co-culture system of Sertoli cells and spermatocytes, to analysis of the association between CD147-MCTs coexistence and activity with intracellular and extracellular lactate concentrations as well as the deterioration or improvement of lead toxicity. (4) to establish an animal model of lead exposure which CD147 function was blocked by anti-CD147 antibody in testis, and further verify the effect of CD147-MCTs mediated lactate shuttle on the improvement of testicular toxicity induced by lead exposure. The findings from this study will provide novel information for prevention and control of the male reproductive toxicity induced by lead.

环境铅污染致雄性精子质量下降是铅毒性研究领域的重要问题，机制不明确。睾丸支持细胞产生的乳酸是精子发生过程中主要能源物质和抗凋亡因子。文献分析和前期实验基础提示铅睾丸细胞毒性涉及干扰细胞间乳酸转运，CD147协同单羧酸转运体（MCTs）介导细胞间乳酸穿梭可能是关键靶环节。本项目拟应用体外睾丸支持细胞-精母细胞非接触式共培养体系，观察低剂量铅暴露后细胞间乳酸水平变化及其与铅致精母细胞损伤的规律；铅对CD147-MCTs复合物共表达和功能的影响与乳酸穿梭的关系；采用调控技术获得CD147和MCTs不同表达水平细胞株，探讨CD147和MCTs表达水平变化对细胞内外乳酸含量的影响以及对铅细胞毒性的恶化或改善作用；构建睾丸局部CD147功能封闭的铅染毒动物模型，进一步在体内水平验证，最终在体内外水平明确CD147-MCTs 介导乳酸穿梭在调节铅暴露睾丸毒性中的作用。为铅生殖毒性防治提供新的靶点和依据。

铅污染致男（雄）性睾丸细胞损伤和精子质量下降严重影响人类的生育力，但其机制仍不明确。睾丸是体内相对独立的微环境，睾丸细胞典型代谢特征是“Warburg”效应。文献分析显示目前仅有少数团队（主要来自美国）在研究外源化学物对睾丸糖代谢影响，研究的主要外源性物质重金属中仅含镉。考虑到环境中铅镉污染常同时存在，体外细胞实验在原有实验设计基础上增加了镉暴露，结果显示铅和镉通过干扰糖酵解或抑制MCT4–CD147转运体表达和共定位减少睾丸支持细胞乳酸产生和运输，两者规律不完成一致。考虑到青春期是睾丸发育最旺盛阶段，整体动物实验铅暴露窗口提前到4周龄小鼠，设置低、中、高3个浓度组和30、60和90天3个观察点；鉴于锌的调节作用，增加高铅+锌干预组。主要结果：高剂量组小鼠血铅含量约100μg／L，相当于中国现行的普通人群血铅水平参考值；铅暴露早期睾丸氧化应激水平较低，葡萄糖摄取、糖酵解和乳酸转运以正向应激调节为主；随着铅暴露时间延长和暴露剂量增加，睾丸抗氧化能力下降，葡萄糖摄取、糖酵解和乳酸转运被显著抑制，可观察到明显的睾丸生精异常改变。相当于青少年补充剂量的锌干预可显著缓解铅诱导的睾丸精子质量损伤；锌可调节铅暴露引起的肠道粘液性质改变，缓解铅所致的肠道菌群结构改变和多样性下降。RNA-Seq转录组测序显示铅暴露组中有288个lncRNA和1202个mRNA表达上调，326个lncRNA和1093个mRNA表达下调，差异表达基因主要富集在MAPK、糖酵解以及脂肪酸合成等通路；有7对lncRNA-mRNA可以形成共表达关系等。本研究模拟内暴露剂量血铅水平控制在卫生监管机构建议的非职业接触人群可接受范围以内，从睾丸糖酵解代谢和精子发生相关基因共同调节的角度阐明了低铅暴露的雄性生殖毒性，有望为部分男性不育症寻找新的分子防治靶点提供依据。另项目执行过程中培养了一批年青学生。