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目的检测ropporin基因在人精子中的表达。探讨Ropporin抗体对人精子运动的影响,评价Ropporin蛋白在精子运动中的功能。方法收集正常男性精子,非连续梯度离心法分离精子,用RT-PCR、免疫印迹和免疫荧光方法检测ropporin基因在精子中的表达和蛋白定位;将纯化精子分别在含不同浓度Ropporin抗体的培养液中孵育(0,0.8,4和20μg/mL),在不同时间点(1,2,6h)用精子计算机辅助分析系统(computer-assisted sperm analysis,CASA)检测精子活动力。结果RT-PCR、免疫印迹和免疫荧光方法均检测出ropporin基因在精子中表达;免疫荧光表明Ropporin主要定位在精子鞭毛的主段和末段;抗体实验显示抗体孵育1h、2h、6h后精子的快速前向运动(a级)和慢速前向运动(b级)均明显受到抑制,并显示剂量依赖性。结论Ropporin在精子鞭毛的主段和末段上的表达与精子的运动密切相关。
Objective To detect the expression of ropporin gene in human sperm. To investigate the effect of Ropporin antibody on human sperm motility and to evaluate the function of Ropporin protein in sperm motility. Methods Sperm were collected from normal male sperm by non-continuous gradient centrifugation. The expression and localization of ropporin gene in sperm were detected by RT-PCR, immunoblotting and immunofluorescence. The purified sperm were cultured in culture medium containing different concentrations of Ropporin antibody (0, 0.8, 4 and 20 μg / mL). Sperm motility was measured by computer-assisted sperm analysis (CASA) at different time points (1, 2, 6 h) Results The expression of ropporin gene was detected in sperm by RT-PCR, Western blotting and immunofluorescence staining. Immunofluorescence showed that Ropporin was mainly located in the main segment and the terminal segment of sperm flagella. Antibody experiments showed that sperm Both fast forward movement (a-stage) and slow forward movement (b-stage) were significantly inhibited and showed dose-dependent. Conclusion The expression of Ropporin in the main segment and the last segment of sperm flagella is closely related to sperm motility.