不同离心方法提取及鉴定精浆外泌体的效果比较

Ultracentrifugation, ExoPerfect^(TM)-MU and PEG6000 for the extraction and identification of seminal plasma exosomes: A comparative study

目的超速离心法、ExoPerfect^(TM)?MU法和PEG6000法比较提取精浆外泌体的应用报道较少。文中旨在比较及鉴定超速离心法,ExoPerfect^(TM)?MU法及PEG6000法提取的精浆外泌体,为外泌体的研究提供不同的选择方法。方法收集2017年3月至7月南方医科大学南方医院检验中心30例正常志愿者的精浆样本。随机平均分成3份,分别采用超速离心法,ExoPerfect^(TM)?MU法及PEG6000法获取提取物,随后采用纳米颗粒跟踪分析仪分析其大小,透射电子显微镜观察其形态,蛋白印迹法检测其典型标志蛋白质。结果超速离心法提取的精浆外泌体中CD63、TSG101含量[(3.29±0.22)、(0.84±0.03)]最多,其次分别是ExoPerfect^(TM)?MU法[(3.25±0.17)、(0.75±0.02)]、PEG6000法[(2.62±0.06)、(0.54±0.10)],差异有统计学意义(P<0.05)。纳米颗粒跟踪分析仪结果提示MODE曲线线性流畅,精浆外泌体直径大小较为集中,粒径峰值平均值分别为105、102、115 nm,差异无统计学意义(P>0.05)。PEG6000法提取精浆外泌体的浓度、电镜下数量[(11.90±1.78)×108/mL、(4.7±1.7)个]较超速离心法[(21.20±0.98)×108/mL、(7.0±1.6)个],ExoPerfect^(TM)?MU法[(19.74±1.45)×108/mL、(6.0±1.63)个]均明显降低(P<0.01);ExoPerfect^(TM)?MU法离心时间[(30±5)min]较超速离心法、PEG6000法[(140±20)、(120±10)min]明显缩短(P<0.001)。结论超速离心法、ExoPerfect^(TM)?MU法、PEG6000法均能成功提取及鉴定精浆外泌体。超速离心法、ExoPerfect^(TM)?MU法外泌体产量较多,ExoPerfect^(TM)?MU法简捷方便。根据不同试验的实际需要,选择合适高效的外泌体提取方法尤为重要。

Objective No studies have been reported on the comparison of ultracentrifugation,ExoPerfect^TM-MU and PEG6000 in extracting seminal plasma exosomes.This article aimed to compare the three methods for the extraction and identification of seminal plasma exosomes.Methods Semen samples were obtained from 30 healthy donors and randomly divided into three portions,followed by extraction of exosomes from the seminal plasma by ultracentrifugation,ExoPerfect^TM-MU,and 8%PEG6000,respectively.The size of the extracted exosomes was measured with the nanoparticle tracking analyzer(NTA),their morphology observed under the transmission electron microscope(TEM),and their protein biomarkers detected by Western blot.Results Significantly higher expressions of CD63 and TSG101 were found in the exosomes extracted by ultracentrifugation than in those extracted by ExoPerfect^TM-MU and 8%PEG6000(P<0.05).NTA revealed a perfect linearity of the MODE curve and insignificant difference in the diameter of the exosomes in the ultracentrifugation,ExoPerfect^TM-MU and 8%PEG6000 groups(105 vs 102 vs 115 nm,P>0.05).Compared with the 8%PEG6000 group,the ultracentrifugation and ExoPerfect^TM-MU groups showed significantly higher concentrations([11.90±1.78]vs[21.20±0.98]and[19.74±1.45]×10^8/mL,P<0.01)and numbers of seminal plasma exosomes under TEM(4.7±1.7 vs 7.0±1.6 and 6.0±1.6,P<0.01).Conclusion Ultracentrifugation,ExoPerfect^TM-MU and 8%PEG6000 are all capable of successful extraction and identification of seminal plasma exosomes,but the former two yield more exosomes,the latter one gives a higher purity,and ExoPerfect^TM-MU is simple and convenient in operation.