规模化制备高纯度牛奶外泌体工艺方法的建立及其分子与细胞生物学活性鉴定

Establishment of process method for large-scale preparation of bovine milk exosomes with high purity and identification of its molecular and cellular biological activities

目的:建立一种规模化的牛奶外泌体纯化工艺,用于实验室制备和工业规模生产,并评价牛奶外泌体的分子与细胞生物学活性。方法:取市售多种全脂或脱脂牛奶,以外泌体提取金标准——超速离心法(UC)作为对照,采用多种化学前处理方法去除乳蛋白,切向流超滤技术(TFF)进一步去除残留的乳蛋白,并实现牛奶样品的初纯与浓缩,多模式色谱法(BE-SEC)精纯牛奶外泌体,采用纳米流式细胞术(NanoFCM)检测牛奶外泌体的粒径、颗粒浓度和纯度,透射电子显微镜(TEM)观察牛奶外泌体的形态表现,高效液相色谱(HPLC)验证牛奶外泌体的尺寸和纯度,Western blotting法检测牛奶外泌体特异性蛋白的表达,基因本体(GO)富集分析差异表达微小RNA(miRNA)的候选靶基因,DAVID Bioinformatics Resources 6.8和KOBAS软件检测京都基因和基因组百科(KEGG)通路中目标候选基因的统计富集度,CytoFlex流式细胞术检测细胞对牛奶外泌体的摄取情况,流式细胞术检测牛奶外泌体作用后Caco-2细胞凋亡率。结果:工艺研究,磷酸钠沉淀法在操作便利性、方法稳定性和产品质量方便均优于其他预处理方法。TFF法可进一步去除残留乳蛋白并可实现液体浓缩目的,有效衔接至下游BE-SEC精纯方法。以该三步工艺所得外泌体样品经TEM成像显示均呈典型的盘状或杯状外泌体形态。NanoFCM法检测的样品粒径为30~150 nm,且优于UC所得外泌体样本纯度。Western blotting法检测,确定外泌体样品含有CD81、Alix和TSG101等外泌体特异性标志物。SEC-HPLC分析,不同批次终产物均为单一均质峰,无游离蛋白污染。GO与和KEGG数据库分析,牛奶外泌体富含免疫相关蛋白与炎症反应调节信号通路相关miRNA。上述制备工艺所得外泌体具备高效穿透细胞膜的能力,4 h内入胞比例达99%。结论:建立了一套由化学沉淀前处理、TFF和BE-SEC三步结合的牛奶外泌体高纯度制备工艺方法。该工艺方法所得牛奶外泌体纯度、产率均优于金标准——UC所得样品。样品理化性质符合典型牛奶外泌体标准,可在数小时内穿透细胞膜,并富含免疫与炎症反应调控相关miRNA。

Objective:To develop a scalable purification process for production of exosomes derived from bovine milk amenable to both laboratory preparation and industrial scale production,and to evaluate the molecular and cellular biological activities of bovine milk exosomes.Methods:A variety of full fat or skim milk samples were taken from the market.The gold standard of exosome extraction,ultracentrifugation(UC),was used as the control.A variety of chemical pretreatment methods were used to remove the milk protein,and tangential flow ultrafiltration(TFF)was used to further remove the residual milk protein;the primary purification and concentration of milk samples were realized;the bovine milk exosomes were purified by biosize exclusion chromatography(BE-SEC);nano flow cytometry(NanoFCM)was used to detect the particle size,concentration and purity of the bovine milk exosomes;the morphology of bovine milk exosomes was observed under transmission electron microscope(TEM);the sizes and purities of the bovine milk exosomes were verified by high performance liquid chromatography(HPLC);the expressions of specific proteins of bovine milk exosomes were detected by Western blotting method;Gene Ontology(GO)enrichment was used to analyze the candidate target genes of differentially expressed microRNA(miRNA);DAVID Bioinformatics Resources 6.8 and KOBAS software were used to detect the statistical enrichment of target candidate genes in Kyoto gene and genome encyclopedia(KEGG)pathway;CytoFlex flow cytometry was used to detect the uptake of bovine milk exosomes;flow cytometry was used to detect the apoptotic rate of Caco-2 cells after treated with bovine milk exosomes.Results:The process development study results showed that the precipitation pretreatment approach with sodium phosphate was superior to all other methods evaluated in regard to feasibility,product quality and reproducibility.TFF method could further remove the residual milk protein and achieved the purpose of liquid concentration,which was effectively linked to the downstream BE-SEC purification method.The exosomes obtained by the three-step process showed the typical disc-shaped or cup-shaped exosomes under TEM.The purity of the sample detected by NanoFCM method was high than that detected by UC,and its partical size was 30-150 nm.The Western blotting results showed that the exosome samples contained CD81,Alix,TSG101 and other exosome specific markers.The SEC-HPLC analysis results showed that the final products of different batches had a single homogeneous peak without free protein pollution.The GO and KEGG database analysis results showed that the milk exosomes were rich in immune related proteins and miRNA related to inflammatory response regulation signaling pathway.The exosomes obtained by the above preparation processes had the ability to penetrate the cell membrane efficiently,and the proportion of exosomes entering the cell within 4 h was 99%.Conclusion:A three-step purification process is established by chemical precipitation pretreatment,TFF and BE-SEC.The purity and yield of bovine milk exosomes obtained by this method are better than those obtained by gold standard—UC.The physicochemical properties of the samples are in line with the standard of typical bovine milk exosomes,which could penetrate the cell membrane within a few hours,and are rich in miRNA related to immune and inflammatory response regulation.