超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞培养上清外泌体的方法学比较

Comparison of Ultracentrifugation and Membrane Based-Affinity Column Methods in Exosome Isolation from Supernatants of Prostate Cancer Cells

目的对超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞上清外泌体优缺点进行比较,为外泌体相关基础实验研究及临床检测提供方法学参考。方法收集前列腺癌细胞上清分别用两种方法提取外泌体,进行电镜鉴定、BCA定量测定蛋白浓度、Western blot检测标志蛋白以及Zetaview颗粒粒径、浓度及电势分析,比较两种方法提取外泌体的优缺点。结果电镜下两种方法均可观察到具有膜结构的典型的类似于茶托状结构的外泌体,但膜亲和柱法提取外泌体背景中有类似蛋白聚合物等杂质的存在,超离背景纯净,每个视野下的外泌体数量要明显多于QIAGEN膜亲和柱法;Western blot测定标志蛋白,根据BCA定量同等上样量超速离心法可观察到明显的β-actin,ALIX,CD63以及EGFR条带,而QIAGEN膜亲和柱法仅可观察到β-actin,ALIX以及EGFR条带,但均较超速离心法条带弱;Zetaview粒径分析,超速离心法粒径分布峰值为116 nm,膜亲和柱法提取外泌体分布峰值为122 nm,均符合外泌体的粒径分布范围;电势分析两者均为负值,符合外泌体的电势分布;而颗粒数与蛋白浓度比值超速离心法提取外泌体要高于QIAGEN膜亲和柱法(t=13.47,P<0.01)。结论超速离心法与膜亲和柱法均可以从细胞上清中提取外泌体,超速离心法步骤繁琐,时间较长,但外泌体纯度高,杂质少,BCA蛋白定量与实际所含外泌体蛋白量基本一致;QIAGEN膜亲和柱法方法简单,可快速从细胞上清中提取到外泌体,但纯度不及超速离心法,BCA蛋白定量比实际所含外泌体蛋白量要高,超速离心法适用于外泌体的各方面研究,而QIAGEN膜亲和柱法由于有杂蛋白的污染,因此更适用于外泌体核酸分析相关研究。

Objective To compare the advantages and disadvantages of ultracentrifugation(UC) and membrane-based affinity column in isolation of exosomes from prostate cancer cell supernatants.Methods Exosomes were isolated by these two methods from LNCaP-AI+F prostate cancer cells,the typical structure of exosomes was observed and captured in electron microscope.BCA assay was used to measure the protein concentration of exosomes,western blot were used to identify the proteins in exosomes and zetaview were used to detect the diameter,particle concentration and zeta potential of exosomes.Results Typical structure of exosomes isolated by two methods could be all observed by electron microscope,while there were some aggregates impurities in the background of exosomes isolated by membrane-based affinity column.There were little impurities in the background of exosomes isolated by UC and more exosomes in a single eyeshot.Western blot results showed that exosomes isolated by UC had obvious β-actin,ALIX,CD63 and EGFR bands while exosomes isolated by membrane-based affinity column only displayed β-actin,ALIX and EGFR bands and were weaker than exosomes isolated by UC.Zetaview particle size analysis showed that the peak number of exosomes isolated by UC and membrane-based affinity column were respectively 116 nm and 122 nm and were both in line with the range of exosome diameter distribution.Zeta Potential of exosomes isolated by these two methods were both negative which accords with the previous reports.The ratio of particle number to protein concentration by UC was higher than that of membrane-based affinity column method(t=13.47,P<0.05).Conclusion Both UC and membrane-based affinity column can extract exosomes from cell supernatants.The UC method is cumbersome and time-consuming,but the exosomes are high in purity and the BCA protein quantification represents the actual exosomes proteins.Membrane-based affinity column method can quickly extract exosomes from cell supernatants,but the purity is lower than ultracentrifugation because of the existence of aggregates proteins.UC is suitable for all aspects of exosome research and membrane-based affinity column is more suitable for exosome nucleic acid analysis because of the contamination of heterologous proteins.