dl-扁桃酸对人精子与阴道黏膜上皮VK2/E6E7细胞的毒性研究

Cytotoxicity of dl-Mandelic Acid on Human Sperm and VK2/E6E7 Cells

关键词:  dl-扁桃酸  壬苯醇醚  VK2/E6E7细胞  细胞毒性

Key words:  dl-mandelic acid  nonoxynol-9  VK2/E6E7 cells  cytotoxicity

摘要:

目的 探讨dl-扁桃酸对人阴道黏膜上皮细胞(VK2/E6E7)与精子的细胞毒性损伤。方法 不同浓度的dl-扁桃酸处理高活力精子20 s，计算机辅助精子分析仪测定精子活率，扫描电子显微镜检测精子受损情况。1.25，2.5 g·L^-1 dl-扁桃酸和4.0 g·L^-1壬苯醇醚(nonoxynol-9，N-9)处理VK2/E6E7细胞，MTT法检测细胞活性，ELISA试剂盒检测乳酸脱氢酶(LDH)释放率，并用流式细胞法检测细胞凋亡率。结果 dl-扁桃酸抑制精子活率的半数有效浓度约为1.25 g·L^-1，最低有效浓度约为2.5 g·L^-1。扫描电镜结果显示，dl-扁桃酸处理组精子表面结构与对照组形态相似，无明显受损特征，N-9组精子严重受损。MTT实验结果表明，1.25 g·L^-1 dl-扁桃酸组细胞存活率>95%，2.5 g·L^-1 dl-扁桃酸组细胞存活率约为50%。1.25，2.5 g·L^-1 dl-扁桃酸处理组LDH释放量只有微量检出，远低于N-9组。流式细胞检测表明，1.25，2.5 g·L^-1 dl-扁桃酸处理组中活细胞和晚期凋亡比例与对照组相近，早期凋亡细胞略少于对照组，死亡细胞稍多于对照组，N-9组大量细胞死亡。结论 dl-扁桃酸对VK2/E6E7细胞毒性远低于N-9，其对精子的抑制作用不是通过破坏精子质膜与鞭毛来实现。

Abstract:

OBJECTIVE To study the cytotoxicity of dl-mandelic acid to VK2/E6E7 cells and human sperm. METHODS The high vigor sperm was incubated with dl-mandelic acid for 20 s, the sperm survival rate was evaluated by computer assistant spermanalysis(CASA). The damage of sperm surface structure was detected by scanning electron microscope. The VK2/E6E7 cells were incubated with 4.0 g?L^-1 nonoxynol-9(N-9) or dl-mandelic acid of 1.25 and 2.5 g?L^-1, the cells survival rate was evaluated by MTT assay, the release rate of LDH was detected by ELISA, and the apoptosis rate were detected by flow cytometry. RESULTS The median effective concentration and the minimal effective concentration of dl-mandelic acid to sperm were 1.25 g?L^-1 and 2.5 g?L^-1. There were no obvious damage characteristics on sperm plasmalemma or flagellum of dl-mandelic acid groups detected by scanning electron microscope, but the sperm plasmalemma or flagellum of N-9 group were severely damaged. The VK2/E6E7 cells survival rate of 1.25 g?L^-1 dl-mandelic acid group was>95%, survival rate of 2.5 g?L^-1 dl-mandelic acid group was about 50%. The LDH release rates of dl-mandelic acid groups were infinitesimal, and far below N-9 group. The living cells and late apoptotic cells of dl-mandelic acid groups were close to control group, showed no significant difference; the early apoptotic cells were little less and dead cells were little more than control group, while most cells of N-9 group were dead. CONCLUSION The dl-mandelic acid has a lower cytotoxicity to VK2/E6E7 cells than N-9. The inhibitory effect of dl-mandelic acid on sperm is not by destroying sperm plasmalemma or flagellum.