牛乳外泌体中miRNA的测序与分析

采用密度梯度离心法提取牛乳外泌体，利用Illumina测序技术对外泌体中非编码小RNA(sRNA)进行测序，探索牛乳microRNA(miRNA)的表达谱。对原始序列进行质量控制，共获得3 899 629条纯净sRNA序列，长度集中于28 nt，与数据库比对后鉴定到61种已知miRNA,346种新miRNA。基因本体论富集结果表明，牛乳外泌体miRNA在细胞过程、单一生物体过程、代谢过程等生物过程发挥作用；主要构成细胞、细胞器等组成；主要参与结合、催化活性、转运蛋白活性等分子功能。京都基因与基因组百科全书通路富集结果表明，已知miRNA与新miRNA靶基因均显著富集在百日咳（ko05133）、趋化因子信号通路（ko04062）、内吞作用（ko04144）、溶酶体（ko04142）等通路，牛乳外泌体miRNA在特定信号通路中发挥重要作用。     

山羊乳及绵羊乳外泌体miRNAs表达谱的分析与差异比较

为揭示山羊乳和绵羊乳外泌体的潜在功能差异，对山羊乳和绵羊乳外泌体进行提取和鉴定，利用Illumina平台对羊乳外泌体中非编码小RNA(sRNA)进行高通量测序，并对微小RNA(miRNA)表达谱和功能进行注释。在山羊乳外泌体中鉴定出161种已知miRNAs序列和5种新型miRNAs序列；绵羊乳外泌体中鉴定出80种已知miRNAs序列和7种新型miRNAs序列，其中miR-148a、miR-26a和let-7b在2种羊乳样本中表达量较高。miR-151只在山羊乳外泌体样本中检出。通路富集发现山羊乳miRNAs靶基因分子功能集中在结合和催化活性等方面，而绵羊乳外泌体miRNAs靶基因具有肠菌素结合的功能，且对宿主发育具有一定促进作用。结果提示山羊乳和绵羊乳来源的miRNAs可能具有某些共性和独特的靶基因类型，可能在缓解病原微生物感染、炎症、抑制癌细胞增殖迁移等方面发挥不同功能。上述miRNAs测序与分析研究可为羊乳外泌体的后续功能开发提供参考。     

不同泌乳期人乳外泌体miRNA表达谱的研究

采用超高速离心法从人乳中提取出囊泡结构物质，从粒径、形态和外秘体特异性标记蛋白3个方面来确定提取的物质为外泌体。采用高通量测序得到不同泌乳期外泌体中microRNA(miRNA)的表达谱。对表达谱进行分析，在人乳外泌体中共检出852种miRNA，有283种miRNA在早、中、晚3个泌乳期中均有所表达。其中，表达量排名前10位的miRNA占到总量的50%以上。在外泌体中高表达的miRNA(占总外泌体miRNA表达量2%以上的)并没有受到泌乳期的显著影响；而在外泌体中低表达的一些miRNA(占总外泌体miRNA表达量0.01%以下的)容易受到泌乳期的显著影响。此外，发现在人乳外泌体中有88种miRNA与炎症相关，其中有65种在不同泌乳期均稳定表达。综上，人乳外泌体可能具有调控炎症的功能，本研究为更好地研究不同泌乳期人乳外泌体生理功能提供基础数据。     

n-3多不饱和脂肪酸对小鼠血液外泌体中miRNA的调节和抑制肥胖作用

目的：研究体内n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids,n-3 PUFAs）含量的增加对小鼠体质量和血液中外泌体miRNAs表达的影响,探讨n-3 PUFAs通过外泌体抑制肥胖的作用机制。方法：利用能自发生成n-3 PUFAs的fat-1转基因小鼠和同窝野生型小鼠（对照）,通过高脂饮食（high-fat diet,HFD）建立肥胖动物实验模型,测定小鼠体质量。提取小鼠血浆中的外泌体并鉴定;分离外泌体内的RNA,构建文库并进行miRNA高通量测序。根据测序结果通过生物信息学方法分析找到其调控的靶基因和相关联的通路,发现miRNA-靶基因互作关系。验证miRNA与肥胖的关联度以及在肥胖中所发挥的作用。结果：fat-1转基因小鼠体质量明显低于野生型;外泌体提取鉴定成功;miRNA高通量测序结果显示,不同小鼠组间进行对比时,差异表达显著（P＜0.05且差异倍数（fold change,FC）≠1）的miRNA有46 个;生物信息学分析发现6 个重要miRNA（mmu-miR-665-3p、mmu-miR-122-5p、mmu-miR-122-3p、mmu-miR-194-5p、mmu-miR-34c-5p、mmu-miR-223-3p）落在脂肪酸代谢通路以及内吞通路关键位置,功能与脂质代谢和肥胖相关,所对应的靶基因分别为Fads1、Elovl2、Elov6、Hadha、Scad1、Scad2、Hsd17b12、Acot2、Acot4和Arf6、H2-T-ps、Arrb1、Ist1、H2-T10、Wwp1、Snx4、IL2rb、Mvb12b、Rab11、fip3、Kif5a、Nedd4l。结论：n-3 PUFAs含量的增加能够有效降低小鼠的体质量,抑制肥胖。n-3 PUFAs能够调节血液中外泌体内miRNA的表达,其中具有显著差异性的miRNA与肥胖有关,其相关的靶基因集中在脂质代谢相关的分子通路中,提示可能是n-3 PUFAs降低体质量抑制肥胖机制之一。     

内源性n-3多不饱和脂肪酸对下丘脑神经干细胞来源外泌体miRNA的调节

目的：通过培养fat-1转基因小鼠下丘脑神经干细胞获取外泌体并提取总RNA进行高通量测序,研究内源性n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids,n-3 PUFAs）对丘脑神经干细胞来源外泌体miRNA的调节作用,探究n-3 PUFAs抑制下丘脑炎症及肥胖的作用机制。方法：分别培养fat-1转基因小鼠与野生型C57BL/6小鼠下丘脑神经干细胞,从细胞培养上清液中收集外泌体并提取总RNA,经高通量测序后利用生物信息学技术对差异表达的miRNA进行分析及靶基因预测,对靶基因进行基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）富集分析后,利用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction,qPCR）进行相对表达量的测定。结果：与野生型C57BL/6小鼠相比,fat-1转基因小鼠下丘脑神经干细胞来源的外泌体miRNA具有特异表达谱,且具有显著差异表达的miRNA靶基因处于下丘脑炎症、脂肪酸代谢等通路中关键位置。经qPCR验证,差异表达的miRNA关键靶基因表达均受到调控。结论：内源性n-3 PUFAs水平的增加可以调节下丘脑神经干细胞外泌体中miRNA的表达,进而调控炎症反应与脂质代谢相关基因的表达,从而发挥抑制下丘脑炎症、抵抗肥胖的作用。     

乳源外泌体的研究进展

乳源外泌体是一种新发现的功能囊泡,直径大约在30~200 nm,为具有膜结构的小囊泡,由动物乳腺上皮细胞分泌。乳源外泌体含有许多蛋白质、脂质、核酸等生物活性物质。最近研究发现乳源外泌体具有介导细胞交流、促进细胞增殖、参与免疫反应等生物学功能,可通过离心、化学、免疫亲和等方法从乳中分离,并对其形态、大小、特异性物质进行鉴定。本文综述了乳源外泌体的生物学功能、分离及鉴定方法、蛋白组学、脂质组学、RNA组学研究,期望对乳源外泌体的研究与开发提供科学参考。     

乳源外泌体的提取及鉴定方法研究进展

乳外泌体是乳中纳米级的胞外囊泡,不仅能够作为药物载体,而且具有促进肠道发育、免疫调节等功能,在婴幼儿配方食品以及功能性食品开发方面具有极大的潜力。外泌体含量低是限制其广泛应用的瓶颈之一。目前,外泌体提取及鉴定的方法尚没有统一的标准,选择合适的乳外泌体提取、鉴定方法对后续的深入研究十分关键。据此,本文综述了目前乳外泌体的提取及鉴定方法,探讨了尚未应用于乳外泌体提取鉴定但理论可行的新方法,期望为乳外泌体的研究与开发提供理论参考。     

高通量测序技术探究驴乳粉对皮肤细胞的影响

基于RNA-seq技术探究驴乳与角质形成细胞和成纤维细胞的相互作用。驴乳分别与角质形成细胞、成纤维细胞共孵育后,采用RNA-seq筛选差异表达基因（DEGs）,进行GO和KEGG富集分析。驴乳作用角质形成细胞后共筛选出819个DEGs,其中420个上调基因,399个下调基因。GO富集分析显示,差异表达基因参与与表皮发育、角质细胞分化、角化等生物学过程。KEGG富集分析显示,驴乳作用角质形成细胞涉及的分子机制包括IL-17信号通路、细胞因子-细胞因子受体相互作用、金黄色葡萄球菌感染等。驴乳作用成纤维细胞后共筛选出507个DEGs,其中298个基因上调,209个基因下调。GO富集分析显示,驴乳参与成纤维细胞的细胞黏附的正调控、脂质定位的调节、脂质转运的调节等生物学过程;KEGG结果显示,驴乳作用成纤维细胞涉及的分子机制包括细胞因子-细胞因子受体相互作用和c GMP-PKG信号通路等。为阐明驴乳对皮肤细胞功能的分子作用机制提供了科学依据。     

乳源外泌体对微生物的作用

外泌体是纳米级(直径40~160 nm)的胞外囊泡,在细胞信号转导、免疫应答和抗原递呈过程中发挥作用,并存在于多种体液,包括血清、唾液、尿液、脑脊液和乳液等。乳外泌体携带蛋白质、miRNA等多种功能分子,很多与人体免疫功能相关。摄取乳液后,乳外泌体中的内容物可被人体肠道吸收,从而发挥健康作用。本文总结国内外有关乳源外泌体与微生物关系的研究报道以及本团队的研究成果,阐述外泌体的生物发生过程,主要组成成分,外泌体与病原微生物感染的关系以及乳外泌体对肠道微生物的作用,为研究乳外泌体对人体的营养和健康功效提供参考。     

驴初乳与常乳乳脂球膜蛋白质组的对比分析

为阐明驴初乳与常乳中乳脂球膜蛋白的差异,利用蛋白质组学技术对二者进行蛋白质组差异分析。在驴初乳和驴常乳中分别鉴定到216 种和215 种乳脂球膜蛋白,并在其中筛选出40 种差异蛋白,包括15 种差异表达蛋白和25 种特异表达蛋白。将驴初乳与常乳中的差异蛋白进行生物信息学分析发现,差异蛋白主要参与的细胞组成为细胞外泌体、胞外囊泡、胞外细胞器等;主要参与的生物过程为对外部刺激的反应过程、细胞增殖过程、血管形态生成过程等;主要参与的分子功能为金属离子结合、阳离子结合、钙离子结合等。此外,这些差异蛋白主要参与补体和凝血级联,为生成IgA而形成的肠道免疫网络等代谢通路。利用蛋白质网络互作分析发现,差异蛋白中存在具有高连接度的关键乳脂球膜蛋白因子。本研究有助了解驴乳脂球膜蛋白的生物学特性,并为驴乳中乳脂球膜蛋白的营养学研究及相关配方乳粉的研发提供理论基础。     

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell–cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.     

Yak milk–derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment

Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal microRNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk–derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk–derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.     

2种不同牛乳外泌体提取方法的比较研究

目的比较研究2种不同的牛乳外泌体提取方法。方法分别采用超速离心法和蔗糖密度梯度离心法从牛乳中提取外泌体,利用透射电镜、动态光散射仪、纳米颗粒追踪仪、免疫印迹等技术对牛乳外泌体进行全面的鉴定表征。结果利用上述2种方法提取获得的牛乳外泌体呈现双层膜的杯托状结构,总体粒径分布在30～200 nm之间且外泌体中常见的特异标记蛋白CD9、CD81、TSG101均存在,不存在污染蛋白Calnexin。结论超速离心法与蔗糖密度梯度离心法均可从牛乳中提取获得结构完整的外泌体,与超速离心法相比,通过蔗糖密度梯度离心法可获取杂质较少、粒径分布更集中的牛乳外泌体,但该法耗时长、外泌体浓度较低。     

模糊数学评价法优化驴乳奶啤稳定性的预处理参数及香气成分分析

基于模糊数学评价法对驴乳奶啤稳定性预处理参数初步优化,结合气相色谱-质谱联用（GC-MS）技术确定驴乳奶啤的最佳预处理参数,通过与牛乳奶啤香气成分差异化对比,探究驴乳奶啤的香气组成。结果表明,最佳预处理参数为单甘脂添加量0.10%、海藻酸丙二醇酯添加量0.15%、果胶添加量0.20%。在此最佳条件下,5号驴乳奶啤综合评分最高（84.20分）,稳定性系数、离心沉淀率、黏度分别为（98.23±0.17）%、（5.21±0.14）%、（37.13±0.08） mPa·s。从牛乳奶啤及5号、9号驴乳奶啤中共检测出79种香气化合物,包括醇类11种、酯类25种、醛类8种、酸类12种、酮类6种、苯类2种、烷烃类12种、萜烯类2种、其他类1种,其中醇类、酯类和酸类挥发性化合物相对含量较高,对牛乳奶啤及5号、9号驴乳奶啤共有的18种香气成分进行主成分分析,经综合评价得出5号驴乳奶啤香气品质最佳,GC-MS分析结果与综合模糊评判结果一致。     

乳中miRNA稳定性与功能研究进展及其作为质量控制指标的分析

microRNAs(miRNAs)是一种长度大约为22个核苷酸的非编码RNA,存在于血浆、血清、唾液、乳汁等体液中,通过调控基因表达参与各种生物过程。乳中miRNA作为新发现的生物活性成分,具有免疫等功能,在反复冻融、酶、低pH等处理条件下也能保持稳定。该文简述了miRNA的合成及功能,详细论述了乳中miRNA的分离提取与稳定性以及母乳和动物乳中miRNA的免疫功能研究进展,分析了乳中miRNA作为潜在的标记物检测乳品质量的可行性,从而为今后以乳中miRNA作为一个新型指标分析检测乳质品质量提供参考,为乳中miRNA的研究提供新的方向。     

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.     

驴乳抑制胰岛素抵抗形成与氨基酸代谢的相关性

目的探讨驴乳(donkeymilk,DM)抑制高脂饮食诱导的胰岛素抵抗形成与氨基酸代谢的相关性。方法将36只C57BL/6N小鼠随机分为空白对照(Con)组,高脂饮食(high fat diet, HFD)组和HFD+驴乳组。给予空白对照组小鼠普通饲料及生理盐水灌胃, HFD组给予高脂饲料及生理盐水灌胃, HFD+驴乳组给予高脂饲料,并且每天按0.1 mL/10 g驴乳灌胃。饮食和驴乳干预8周后,检测各组小鼠血糖血脂水平和胰岛素敏感性,采用超高效液相色谱-串联质谱测定小鼠血浆靶标氨基酸含量,结合SIMCA-P软件对氨基酸数据进行PLS-DA分析,并分析变化的靶标氨基酸与血糖血脂水平的相关性。结果高脂饮食诱导8周,与Con组小鼠相比,HFD组小鼠体重、随机血糖(glucose, GLU)、总胆固醇(total cholesterol, TC)水平、口服葡萄糖耐量(oral glucose tolerancetest,OGTT)曲线下面积、胰岛素耐量曲线下面积和空腹血清胰岛素水平显著升高(P0.05),胰岛素敏感指数下降(P0.05)。与HFD组相比, HFD+驴乳组体重、GLU、TC和甘油三酯(triglyceride, TG)水平、OGTT曲线下面积显著降低(P0.05)。与空白对照组小鼠比较,HFD组小鼠血清中丙氨酸、半胱氨酸、脯氨酸、甲硫氨酸含量升高,赖氨酸含量降低(P0.05);与HFD组比较,HFD+驴乳组中的半胱氨酸含量显著降低(P0.01)。PLS-DA分析结果显示驴乳给予后小鼠血清氨基酸代谢谱较模型组发生了显著变化。Pearson相关分析显示,脯氨酸、甲硫氨酸和半胱氨酸分别与空腹血糖(fasting blood glucose, FBG)、GLU、TC水平呈显著性正相关(P0.05)。结论驴乳能够延缓高脂饮食诱导小鼠胰岛素抵抗的发生,并且驴乳能够影响胰岛素抵抗小鼠的血清氨基酸代谢谱,被改变的半胱氨酸、脯氨酸、甲硫氨酸的代谢与胰岛素抵抗的发生发展密切相关。     

Yak milk–derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation

Intestinal epithelial cells (IEC) are important parts of the mucosal barrier, whose function can be impaired upon various injury factors such as lipopolysaccharide. Although food-derived exosomes are preventable against intestinal barrier injuries, there have been few studies on the effect of yak milk–derived exosomes and the underlying mechanism that remains poorly understood. This study aimed to characterize the effect of exosomal proteins derived from yak and cow milk on the barrier function of IEC-6 treated with lipopolysaccharide and the relevant mechanism involved. Proteomics study revealed 392 differentially expressed proteins, with 58 higher expressed and 334 lower expressed in yak milk–derived exosomes than those in cow exosomes. Additionally, the top 20 proteins with a relatively consistent higher expression in yak milk exosomes than cow milk exosomes were identified. Protein CD46 was found to be a regulator for alleviating inflammatory injury of IEC-6. In vitro assay of the role of yak milk exosomes on survival of IEC-6 in inflammation by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay confirmed the effectiveness of yak milk exosomes to increase IEC-6 survival up to 18% for 12 h compared with cow milk exosomes (up to 12%), indicating a therapeutic effect of yak milk exosomes in the prevention of intestinal inflammation. Furthermore, yak and cow milk exosomes were shown to activate the PI3K/AKT/C3 signaling pathway, thus promoting IEC-6 survival. Our findings demonstrated an important relationship between yak and cow milk exosomes and intestinal inflammation, facilitating further understanding of the mechanisms of inflammation-driven epithelial homeostasis. Interestingly, compared with cow milk exosomes, yak milk exosomes activated the PI3K/AKT/C3 signaling pathway more to lower the incidence and severity of intestine inflammation, which might represent a potential innovative therapeutic option for intestinal inflammation.