睾丸注射白消安后SSCs和SCs的毒性调控关系研究

The technology of receipient preparation for spermatogonial stem cells (SSCs) plays an important role in improving the fecundity of male animals. However, the high mortality rate caused by the common method of intraperitoneal injection of busulfan has not been solved yet. The key of SSCs receptor preparation is to improve the preparation efficiency and avoid the death of recipient. In our previous study, we successfully prepared recipients for SSCs transplantation by testicular injection, and obtained donor offspring and avoid the death of the recipient. It was found that the exhaustion and recovery of SSCs synchronized with that of sertoli cells (SCs) and the regulation of SCs and SSCs affected the exhaustion and recovery of SSCs. To investigate the regulation relationship among SCs, SSCs and series spermatogenic cells to overcome the toxicity of busulfan, we will carry out the separate culture and co-culture of SSCs and SCs and culture of seminiferous tubules from mouse and porcine. The cultured cells and the seminiferous tubules are treated with different dosages of busulfan. The key genes upstream and downstream of regulatory pathway of the apoptosis, proliferation and differentiation will be checked to explore the mechanism and path of toxicity of busulfan. The findings of this research will help to break through the bottleneck of efficiency and safety of receptor preparation technology for SSCs transplantation and promote rapid expansion of excellent breeding livestock by improving the fecundity of major male animals.

精原干细胞（Spermatogonial stem cells, SSCs）移植受体制备，对充分挖掘种公畜繁殖力意义重大，但常用白消安腹腔注射法的高死亡率至今无法解决。避免受体死亡，提高效率，是SSCs受体制备技术研发的关键。本团队前期通过睾丸注射成功制备SSCs移植受体，获得了供体后代，避免了受体死亡，并发现SSCs消亡与恢复伴随了支持细胞（Sertoli cells, SCs）消亡与恢复，结合前人研究发现，SCs与SSCs的调控关系影响了SSCs消亡与恢复。为解决SCs与SSCs和系列生精细胞的白消安毒性调控关系科学问题，申请人拟通过小鼠和猪SSCs、SCs单独与共培养及曲细精管培养，分不同时间点检测分析白消安处理后细胞凋亡、增殖、分化调控通路上下游关键基因，探明白消安毒性作用机制与路径，突破受体制备技术效率与安全瓶颈，促进顶级公畜繁殖潜力充分挖掘和良种家畜快速扩繁。

白消安因为对精原干细胞（SSCs）及系列生精细胞的毒害作用，而被用于SSCs移植受体制备，本团队前期应用睾丸注射法避免了腹腔注射白消安导致的处理鼠高死亡现象表明，深入揭示其毒性机理，才能科学使用白消安并降低甚至避免其机体毒害作用。为此，本团队进行了SSCs和支持细胞（SCs）的体外培养和白消安毒害实验，发现体外培养的SCs和SSCs经白消安处理后，细胞逐渐萎缩，增殖受阻，数量变少。为进一步揭示体内各种细胞相互协调情况下，白消安毒害发生的生理机制，对小鼠睾丸进行白消安注射，注射鼠睾丸逐渐失重，精子生成数量逐渐减少，生物素示踪、高效液相靶向测定和免疫定位研究发现，白消安可快速通过BTB，使SSCs和SCs间及SCs间的紧密连接蛋白Occludin和缝隙连接蛋白connexin43在白消安注射后逐渐减少，影响了SSCs、SCs等细胞间相互支撑的物理连接，并损害了各类细胞间的信息传递系统，也破坏了曲细精管BTB的结构、功能。损伤BTB破坏了精子发生的龛环境，不利于精子发生；物理连接的损伤降低了SSCs和SCs的生活力和相互调节能力，并使各类细胞逐渐萎缩，更重要的是生精细胞丧失了SCs的调节与营养供应，并从SCs上脱落和走向衰亡。另外，本实验还发现，白消安注射后，血清TNF-α、IL-1β和IL-6等炎症因子的浓度逐渐上升，推测白消安注射导致了睾丸炎症反应，而睾丸炎症反应如何在损害细胞间连接，阻碍精子发生中发挥作用，还有待于进一步深入研究。该机制的揭示，将对如何从炎症发生规律入手保护雄性动物生精机能，具有一定的理论与实践意义。