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

作　　者：舒妮燕 于合国[2] 王玉柱[2] 李卫华[2] 施惠娟[2] 刁华[2] SHU Ni-yan YU He-guo WANG Yu-zhu LI Wei-hua SHI Hui-juan DIAO Hua(Department of Reproductive Biology, Shanghai Medical College, Shanghai 200032, China Key Lab of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai 200032, China)

机构地区：[1]复旦大学上海医学院生殖生物学研究室,上海200032 [2]复旦大学生殖与发育研究院,上海市计划生育科学研究所,国家人口和计划生育委员会计划生育药具重点实验室,上海200032

出　　处：《复旦学报（医学版）》2017年第4期395-402,共8页Fudan University Journal of Medical Sciences

基　　金：国家重点研发计划(2016YFC1000905);国家自然科学基金面上项目(81671508);上海市科技创新行动计划项目(15431903000)~~

摘　　要：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.

关 键 词：色瑞替尼 精子制动 避孕潜能 小鼠 

分 类 号：R697[医药卫生—泌尿科学]