B7-H3表达缺陷在特发性少、弱、畸精症发病中的作用机制研究

The microenvironment around sperm is crucial for spermatogenesis, spermmaturation and its normal functions. Previous studies showed that B7-H3 is significantly highly expressed in testis, epididymis, seminal vesicles and prostate. However, the role of biology of B7-H3 on spermatogenesis and spermmaturation remains unknown. Our recent studies indicated that seminal B7-H3 levels in patients with idiopathic oligozoospermia, asthenozoospermia and teratozoospermia were significantly lower than that of healthy donors. Additionally, B7-H3 can induce cell differentiation, promote sperm motility and enhance cell adhesion. According to our findings and previous reports, we hypothesizes that B7-H3 plays a critical role in the spermatogenesis and spermmaturation, and its expression defect induces the pathogenesis of idiopathic oligozoospermia, asthenozoospermia and teratozoospermia. To test this hypothesis, ELISA, sperm function test, immunohistochemisty, Western-blot, microinjection into seminiferous tubules, RNAi were performed to investigate: 1) the association between seminal B7-H3 and sperm quality and its clinical significance; 2) the effect of B7-H3 on sperm functions; 3) the expression levels, distribution and trend in testis and epididymis; 4) the effect of expression defect of B7-H3 on spermatogenesis, spermmaturation and sperm functions. This project aims to analyze the effect and mechanism of B7-H3 expression defect on idiopathic oligozoospermia, asthenozoospermia and teratozoospermia, in order to provide new ways for future biological treatment.

研究表明，B7-H3在睾丸、附睾、精囊及前列腺中均高表达，但其是否影响精子的发生、成熟和功能目前尚无报道。我们前期研究发现，特发性少、弱、畸精症患者精浆中B7-H3水平显著低于健康志愿者，且该分子可以诱导细胞分化、促进精子活力、促进细胞间粘附。据此我们推测B7-H3可能参与了精子的发生和成熟过程，其表达缺陷可能导致了特发性少、弱、畸精症的发生。鉴此，本项目拟通过ELISA检测、精子功能学实验、免疫组化、Western-blot、曲细精管及附睾内显微注射、RNA干扰、动物模型构建等手段，从临床、分子、细胞、组织和动物水平多层次研究以1）分析精浆B7-H3与精子质量之间的关系及临床意义；2）明确B7-H3对精子功能的影响；3）确定B7-H3在睾丸和附睾中的表达水平、分布情况及表达趋势；4）揭示B7-H3表达缺陷对精子发生、成熟和功能的影响。探索特发性少、弱、畸精症的发病机制，为治疗提供新思路。

本项目首先分析了人精浆B7-H3与不育和精液参数之间的关系。结果显示特发性少精、弱精、少-弱及弱-畸精子症患者的精浆B7-H3水平显著低于健康男性。且精浆B7-H3水平与精子密度、活率、活力等参数呈显著负相关。为了明确精浆B7-H3是否对成熟精子功能有所影响，而后我们研究了B7-H3在体外对不育患者精子功能的影响。结果显示B7-H3可以提高少、弱及少-弱精症患者的精子活力，但对精子获能和顶体反应无明显影响。为了进一步探索B7-H3是否可能及如何影响精子的发育，我们分析了不同性成熟阶段小鼠睾丸及附睾组织内B7-H3的表达分布规律及变化趋势。结果显示，小鼠附睾中以体部表达量最高，主要表达与附睾管上皮细胞胞质及包膜。睾丸中则表达于支持细胞胞质及包膜上。随性成熟（4月龄）达到顶峰后逐渐下降。B7-H3在生殖腺中的这种分布规律提示其可能参与了精子的发生和成熟过程。因此，我们提取了小鼠精原干细胞（SSCs），并进一步研究了B7-H3对SSCs增殖和周期的作用。我们发现B7-H3蛋白可以促进SSCs的增殖，加速细胞周期。而这是依赖于SSCs胞内PI3K信号传导的。这说明小鼠睾丸和附睾中的B7-H3可能参与了精子发育和成熟的过程。基于临床样本和小鼠模型的研究结果，我们认为睾丸和附睾中的B7-H3可能参与了精子的发生和发育过程，这位进一步了解特发性少、弱、畸精子症的发生机制及临床治疗提供了新思路。