色瑞替尼对精子制动作用和质膜损伤的体外研究

The spermostatic and plasma membrane injury effects of ceritinib in vitro

目的研究前期筛选到的精子功能调节候选化合物色瑞替尼的体外精子制动作用和机制。方法计算机辅助精液分析(computer assistant sperm analysis,CASA)系统测定色瑞替尼对人和小鼠高活力精子20 s内的制动效果;低渗膨胀(hypo-osmotic swelling,HOS)实验、SYBR-14/PI染色探究精子质膜的完整性;用电镜观察色瑞替尼处理后精子质膜的变化;用色瑞替尼处理VK2/E6E7、End1/E6E7、Ect1/E6E7细胞,CCK-8及荧光染色法检测细胞活性。结果色瑞替尼对于(5~10)×106/mL人精子的20 s瞬间杀精最小有效浓度(minimal effective concentration,MEC)为(74.87±31.46)μmol/L,而相同条件下测到的杀精药物壬苯醇醚(nonoxynol-9,N-9)的MEC为(219.75±20.89)μmol/L。CASA系统分析表明,色瑞替尼对精子的制动作用有时间-浓度依赖关系。HOS实验及SYBR-14/PI染色法验证色瑞替尼对精子质膜有一定的损伤,并可致精子死亡。电镜显示色瑞替尼处理后的精子质膜破裂,头尾分离较多。色瑞替尼对上皮细胞有一定损伤。结论色瑞替尼对精子有快速的制动作用,具有潜在的避孕药效。

Objective To explore the in vitro spermostatic effects and the mechanisms of ceritinib, a novel candidate from the active compound pools previously screened for the regulation of sperm function. Methods The vigor sperm of human and mouse were incubated with ceritinib for 20 seconds, and the sperm motility was evaluated by computer assistant sperm analysis （CASA）. The integrity of the sperm plasma membrane and the survival ratio of sperm was assessed by hypo-osmotic swelling （HOS） assay and SYBR-14/PI staining. The damage of sperm plasma membrane was detected by electron microscope. The cytotoxicity of ceritinib to VK2/E6E7, End1/E6E7 and Ect1/E6E7 cells was measured by CCK-8 assay and fluorescent staining. Results The minimal effective concentration （MEC） of ceritinib to （5-10）×10^6/mL human sperm within 20 seconds was （74.87±31.46） μmol/L, which was significantly lower than the MEC of nonoxynol-9 （219.75±20.89） μmol/L measured in the same condition. The time-dose related spermostatic effects of ceritinib were measured by CASA system. Ceritinib was able to damage sperm membrane and caused sperm death with HOS and SYBR-14/PI staining. With electron microscopy, the sperm membrane was observed to be ruptured, and the heads and tails of sperm were separated by ceritinib. Ceritinib was also able to damage the epithelial cells. Conclusions Ceritinib has acute spermostatic effects and potential contraceptive effects.