实时定量PCR检测Kif2amRNA在男性精液中的表达

Detection of Kif2a mRNA in male ejaculate by real-time fluorescence quantitative RT-PCR

目的检测驱动蛋白重链成员2A(Kif2a)在男性精液中的表达,探讨Kif2a在不育男性精液中表达的意义。方法收集72例新鲜精液样本液化,经过离心等预处理后用TRIzol试剂提取精液RNA并逆转录成cDNA。运用实时荧光定量PCR检测精液中总Kif2a mRNA的量,并选取管家基因甘油醛-3-磷酸脱氢酶(GAPDH)作为内参对照。样本依据精液临床检测指标分为异常组和正常组。结果实时定量PCR法检测所得结果显示Kif2a mRNA存在于人类精液中。对Kif2a mRNA进行相对定量分析显示,在精子存活率、精子密度、精子活动率(a+b+c级精子)方面,异常组中Kif2a mRNA的表达量较正常组明显降低,差异有统计学意义(P<0.05)。结论结合Kif2a在男性异常和正常精液中的差异性表达,其在精子的生成和受孕方面是否存在潜在的影响值得进一步探索。

Objective To detect the expression of kinesin heavy chain member 2A （Kif2a） in human ejaculate and discuss the significance of Kif2a in ejaculate of infertile man. Methods Human ejaculate samples were obtained from 72 subjects, sperm RNA was extracted from ejaculate cells by using TRIzol reagent after liquefaction and centrifugation, and the RNA was used for reverse transcription into cDNA. The amount of Kif2a mRNA was quantified by utilizing real-time quantitative PCR （RT-PCR） ; Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） was selected as housekeeping gene. The subjects were grouped as abnormal and normal based on the clinical diagnosis of the ejaculate. Results The results of RT-PCR showed that the Kif2a mRNA is present in human ejaculates. Conjunction with clinical data of sperm vitality, concentration and total motility （ progressive ＋ nonprogressive）, the expression of Kif2a mRNA in abnormal groups was lower than that in normal groups, with the difference between them being statistically significant（P 〈0. 05）. Conclusion According to the distinct expression of Kif2a mRNA in abnormal and normal ejaculate, whether the difference has potential impact on spermatogenesis and fertilization should be studied further.