神经病靶酯酶在三邻甲苯磷酸酯诱导的大鼠精原干细胞自噬中的作用及机制研究

Tri-ortho -cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame-retardants in the industry. It has been shown that TOCP can inhibit the activity of neuropathy target esterase (NTE) and decrease cauda epididymal sperm density, suggesting that NTE might play a role in spermatogenesis. Spermatogenesis is a complex and highly organized process, in which undifferentiated spermatogonial stem cells proliferate and sequentially differentiate, resulting in generation of functional sperm in the testis. Our preliminary results found that TOCP could induce autophagy of rat spermatogonial stem cells. Inasmuch as cAMP/PKA can promote expression of NTE mRNA in HeLa cells and inhibition of cAMP/PKA induces autophagy, we propose that TOCP might inhibit NTE activity and induce autophagic cell death of rat spermatogonial stem cells via inhibition of cAMP/PKA signal. Therefore, we will test this hypothesis by the following approaches: (1) we will employ transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining, a speci?c autophagolysosome marker, and Western blotting to investigate whether TOCP induces autophagic cell death of rat spermatogonial stem cells, (2) phenyl valerate hydrolysis, Western blotting and radioimmunoassay (RIA) will be used to assess the effect of TOCP on NTE activity and cAMP/PKA in these cells and (3), we will determine whether TOCP inhibits NTE activity and induces autophagic cell death of rat spermatogonial stem cells via inhibition of cAMP/PKA signal. The present study will provide a novel insight into the mechanism for the regulation of spermatogonial stem cells.

三邻甲苯磷酸酯（TOCP）常作为增塑剂、软化剂及阻燃剂等，在工业中使用非常广泛。研究显示，TOCP除抑制神经病靶酯酶（NTE）活性外，还抑制精子发生，提示NTE很可能参与精子发生。精原干细胞是精子发生的起始，在基因的调控下有序地增殖并分化为成熟的精子。我们的初步结果发现，TOCP可诱导精原干细胞自噬；鉴于cAMP/PKA被抑制后可导致NTE活性下降，且可诱导细胞自噬；提示TOCP可能通过阻断精原干细胞内cAMP/PKA信号而抑制NTE活性，从而诱导细胞自噬性死亡。本课题通过分离的大鼠精原干细胞，首先用透射电镜、MDC染色和免疫印迹等方法确定TOCP诱导精原干细胞自噬，然后用戊酸苯酯水解法、免疫印迹及放免法等方法确定TOCP对细胞内NTE活性及cAMP/PKA的影响，并最终确定TOCP-cAMP/PKA-NTE-细胞自噬性死亡的线性关系。为深入探讨精原干细胞的调控机理提供理论基础和实验依据。

三邻甲苯磷酸酯（TOCP）常作为增塑剂、软化剂及阻燃剂等，在工业中使用非常广泛。研究显示，TOCP 可抑制神经病靶酯酶（NTE）活性并抑制精子发生，提示NTE 很可能参与精子发生。该课题通过分离的大鼠精原干细胞，发现TOCP可以抑制细胞活性，通过用western blot检测自噬蛋白LC3、Beclin1和Atg5的表达以及用电镜检测细胞内自噬泡情况，证实TOCP可诱导精原干细胞自噬，但对细胞周期和凋亡没有影响。免疫组织化学显示NTE在大鼠精原干细胞中表达，并且TOCP可抑制精原干细胞内NTE活性及其mRNA表达，并可抑制细胞内cAMP水平；然而改变细胞内cAMP水平，并没有明显诱导精原干细胞自噬。由于精原干细胞内高表达细胞色素P450可降解TOCP为活性更高的SCOTP，为此，我们合成了SCOTP，并处理大鼠精原干细胞，我们发现浓度更低的SCOTP可诱导细胞自噬。本课题的研究为深入探讨TOCP对男性生殖毒性机理提供理论基础和实验依据。