过滤对超速离心法提取外泌体的影响

Effect of filtration on exosome extraction by ultracentrifugation

目的明确过滤对超速离心法提取外泌体数量及纯度的影响,为当前外泌体的提取方法提供一定参考。方法收集健康人血浆和肺癌PC-9/IR细胞培养上清,分为未过滤组(unfiltration group,NC)和过滤组(filtration group,GL),过滤组采用0.22μm过滤器过滤。超速离心法提取外泌体,纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)检测过滤前后纳米颗粒(外泌体)数量及粒径大小变化,Aminis成像流式检测过滤前后CD9^(+)外泌体(CD9^(+)exosomes,CD9^(+)Exo)和CD63^(+)外泌体(CD63^(+)exosomes,CD63^(+)Exo)数量及百分比。结果NTA结果显示,与NC组相比,血浆过滤后外泌体数量下降4.7倍,培养上清过滤后外泌体数量下降2.5倍。Aminis成像流式结果显示,血浆中CD9^(+)Exo和CD63^(+)Exo的数量过滤后分别下降了5倍和2.6倍,培养上清中CD9^(+)Exo和CD63^(+)Exo的数量过滤后分别下降了3倍和2.3倍,差异均有统计学意义(P<0.05)。同时,Aminis成像流式结果显示,过滤后颗粒中CD9^(+)Exo和CD63^(+)Exo的百分比没有提高,差异无统计学意义。结论过滤降低了超速离心法提取外泌体的数量,并未提高外泌体的纯度。

Objective To clarify the effect of filtration on the quantity and purity of exosomes extracted by ultracentrifugation,and to provide a reference for the current extraction methods of exosomes.Methods Healthy human plasma and lung cancer cell PC-9/IR cell culture supernatants were collected and divided into unfiltration group(NC)and filtration group(GL),the filtration group was filtered with 0.22μm filter.Exosomes were extracted by ultracentrifugation.The number and size of nanoparticles(exosomes)before and after filtration were detected by nanoparticle tracking analysis(NTA).The number and percentage of CD9^(+)exosomes(CD9^(+)Exo)and CD63^(+)exosomes(CD63^(+)Exo)before and after filtration were detected by Aminis imaging flow cytometry.Results The results of NTA showed that compared with the NC group,the number of exosomes decreased by 4.7 times after plasma filtration,and the number of exosomes decreased by 2.5 times after filtration of the culture supernatant.The results of flow cytometry showed that the number of CD9^(+)Exo and CD63^(+)Exo in plasma decreased by 5 and 2.6 times respectively after filtration,and the number of CD9^(+)Exo and CD63^(+)Exo in culture supernatant decreased by 3 and 2.3 times respectively after filtration.The difference was statistically significant(P<0.05).But the percentage of CD9^(+)Exo and CD63^(+)Exo did not significantly increase after filtered both in plasma and supernatant(P>0.05).Conclusion Filtration decrease the number of exosomes,but not improve the purity of exosomes.