【作者】 李颖；

【导师】 李艾黎；

【作者基本信息】 东北农业大学 ， 食品科学， 2021， 硕士

【摘要】 外泌体是由细胞分泌到胞外的一种囊状小泡,能够将其所携带的内容物传递至靶细胞发挥特定作用。外泌体作为牛奶中重要的生物活性成分,已被证实具有调控自身免疫、预防炎症、促进肠道发育等诸多生理功能。其中,微小核糖核酸(micro RNAs,mi RNAs)被认为是乳源外泌体中的重要功能执行者。以往研究多关注不同乳制品的组成和功能成分的差异,但迄今鲜见关于不同乳制品中外泌体生物学作用的研究。因此,本课题以母乳、鲜牛乳为对照,首先比较不同牛乳制品(超高温灭菌(Ultra-high temperature instantaneous sterilization,UHT)乳、冻干粉和配方粉)中外泌体的形态特征和表面标志蛋白的差异。进而构建正常和脂多糖(Lipopolysaccharide,LPS)诱导RAW264.7巨噬细胞模型,观察不同牛乳制品中外泌体刺激免疫细胞表达mi RNAs、分泌细胞因子和介导信号通路的影响,旨在为我国乳源外泌体的研究与开发提供一定理论依据。一、不同牛乳制品中外泌体的表征研究首先采用马尔文粒径分析仪、透射电镜和蛋白印迹比较不同牛乳制品中外泌体的表征差异。结果表明,提取各组外泌体的峰值均只有一个,在100-150 nm之间;各组外泌体均为圆形或椭圆形,呈脂质双层膜结构。可见从母乳、鲜牛乳及UHT乳、冻干粉和配方粉中均能提取符合标准的外泌体。其中,母乳外泌体混合液背景最干净,牛乳组的外泌体混合液中相对含有较多背景杂质,外泌体颗粒易结团,特别是配方粉外泌体电镜图片中有较多杂质蛋白融合现象。此外,鲜牛乳外泌体表面标志蛋白CD63、TSG101和CD9条带略深而粗,UHT乳和冻干粉组外泌体条带略浅,配方粉组外泌体表面标志蛋白条带最浅。二、不同牛乳制品中外泌体调节巨噬细胞mi RNA表达的研究建立正常状态及LPS诱导的巨噬细胞模型,分析母乳、鲜牛乳、UHT乳、冻干粉和配方粉中提取的外泌体对巨噬细胞表达mi RNAs的影响。结果显示,各外泌体组显著促进了正常巨噬细胞表达免疫相关mi RNAs,如mi RNA-223、mi R-181a和mi R-155,但对mi R-30d没有影响。其中,母乳组中的mi R-155和mi R-223表达高于鲜牛乳组,mi R-181a水平略低于鲜牛乳组,但不具有显著性;鲜牛乳组的mi R-181a表达与冻干粉组差异不显著,但显著高于UHT乳组和配方粉组(P<0.05)。鲜牛乳组与各牛乳制品组mi R-223和mi R-155表达均无显著性差异(P>0.05)。而与LPS诱导的巨噬细胞共培养后,各外泌体组呈现促进mi R-223、mi R-181a,抑制mi R-155过表达的作用。其中,母乳组提高mi R-223,降低mi R-155的表达显著高于其他组(P<0.05);鲜牛乳组促进mi R-181a表达显著高于其他组(P<0.05),抑制mi R-155过表达的作用与冻干粉组相似,显著高于UHT乳组和配方粉组(P<0.05)。三、不同牛乳制品中外泌体激活免疫的研究利用母乳、鲜牛乳、UHT乳、冻干粉和配方粉中的外泌体刺激正常巨噬细胞,探究不同牛乳制品中外泌体对激活巨噬细胞的影响。结果显示:各外泌体组均可激活巨噬细胞,包括促进细胞增殖,增加NO和IL-1β、TNF-α、IL-6的分泌。其中,鲜牛乳和母乳中的外泌体均展示出明显的免疫激活作用;而鲜牛乳组促进IL-1β的作用显著高于各牛乳制品组(P<0.05);促进TNF-α和IL-6的作用与冻干粉组差异不显著,优于UHT乳组和配方粉组。四、不同牛乳制品中外泌体抑制LPS介导炎症的研究建立LPS诱导的巨噬细胞炎症模型,探究不同牛乳制品中外泌体对抑制炎症反应的影响。结果显示,母乳外泌体组抑制LPS诱导的巨噬细胞内ROS积累和促炎因子水平升高的效果最好。鲜牛乳组外泌体抑制NO和IL-6的产生,下调IL-1βm RNA表达的效果显著高于各牛乳制品组。且鲜牛乳组和冻干粉组降低IL-6和TNF-α转录水平的能力优于UHT乳组和配方粉组。鲜牛乳组与冻干粉组和UHT乳组ROS的产生差异不显著,但显著低于配方粉组(P<0.05)。此外,各组外泌体还能不同程度降低LPS诱导的TLR4/NF-κB信号通路的表达。母乳、鲜牛乳、冻干粉组和UHT乳组抑制TLR4受体的蛋白表达无统计性差异,显著高于配方粉组。鲜牛乳组抑制p65和IκBα蛋白磷酸化的作用显著低于母乳组。且鲜牛乳组p-p65和p-IκBα的蛋白表达量与冻干粉组相似,显著高于UHT乳组。配方粉组抑制p65和IκBα蛋白的磷酸化的作用最差。以上结果表明,从母乳、鲜牛乳、UHT乳、冻干粉和配方粉中提取的外泌体均具有免疫系统的双向调节作用,即上调正常巨噬细胞的免疫活性,下调LPS诱导巨噬细胞的炎症反应。且鲜牛乳组的免疫调节作用与冻干粉组相似,高于UHT乳组,配方粉组的免疫调节作用最低。这可能与各组外泌体调节巨噬细胞内表达免疫相关的mi RNAs水平不同有关。更多还原

【Abstract】 Exosome is an extracellular vesicle secreted by cells,which can deliver its contents to target cells to play a specific role.As an important bioactive ingredient in milk,exosomes have been proved to have many physiological functions,such as regulating autoimmunity,preventing inflammation,and promoting intestinal development.Among them,micro RNAs(mi RNAs)are considered to be important functional performers in milk derived exosomes.Previous studies focused on the differences in the composition and functional components of different dairy products,but so far there have been few studies on the biological effects of exosomes in different dairy products.Therefore,in this study,using breast milk and fresh milk as controls,the morphological characteristics and surface marker protein of exosomes from different milk products(ultra-high temperature sterilization(UHT)milk,freezed-dried powder and formula powder)were firstly compared.Furthermore,normal and Lipopolysaccharide(LPS)induced RAW264.7macrophage model was constructed to observe the effect of different milk products exosomes stimulating the expression of mi RNAs,secreting cytokines and mediating signaling pathways of immune cells,in order to provide a theoretical basis for the research and development of milk exosomes in China.1.Characterization of exosomes in different dairy productsFirstly,the characterization differences of exosomes in different milk products were compared by Malvin particle size analyzer,transmission electron microscope and western blot.The results showed that the peak value of exosomes in each extraction group was only one,which was between 100-150 nm.The exosomes in each group were round or oval with lipid bilayer structure.It can be seen that the standard exosomes can be extracted from breast milk,fresh cow milk,UHT milk,freeze-dried powder and formula powder.Among them,the breast milk exosome mixture had the cleanest background,and the exosome mixture in the milk group contained relatively more background impurities,and exosome particles were easy to aggregate,especially the electron microscope images of formula powder exosome showed more impurity protein fusion.In addition,fresh milk exosome surface marker protein CD63,TSG101 and CD9 bands were slightly deeper and thicker,UHT milk and lyophilized powder group exosome surface marker protein bands were slightly shallower,formula powder group exosome surface marker protein bands were the shallowest.2.Studies on the regulation of mi RNA expression in macrophages by exosomes from different milk productsNormal and LPS-induced macrophage models were established to analyze the effects of exosomes extracted from human milk,fresh cow milk,UHT milk,freeze-dried powder and formula powder on the expression of mi RNAs in macrophages.The results showed that each exosome group significantly promoted the expression of immune-related mi RNAs,such as mi RNA-223,mi R-181 a,and mi R-155,in normal macrophages,but had no effect on mi R-30 d.The expressions of mi R-155 and mi R-223 in the breast milk group were higher than those in the fresh milk group,and the level of mi R-181 a was slightly lower than that in the fresh milk group,but there was no significant difference.The expression of mi R-181 a in fresh milk group was not significantly different from that in lyophilized powder group,but was significantly higher than that in UHT milk group and formula powder group(P<0.05).There was no significant difference in the expression of mi R-223 and mi R-155 between fresh milk group and milk product group(P>0.05).However,after co-culture with LPS-induced macrophages,each exosome group showed the role of promoting mi R-223 and mi R-181 a,and inhibiting the overexpression of mi R-155.Mi R-223 and mi R-155 expression were increased and decreased significantly in the breast-feeding group compared with the other groups(P<0.05);the expression of mi R-181 a in fresh milk group was significantly higher than that in other groups(P<0.05),inhibiting mi R-155 overexpression was similar to that of the lyophilized powder group,and significantly higher than that of the UHT milk group and formula powder group(P<0.05).3.Studies on the activation of immunity by exosomes from different dairy productsExosomes in breast milk,fresh milk,UHT milk,freeze-dried powder and formula powder were used to stimulate normal macrophages to investigate the effects of exosomes in different milk products on macrophage activation.The results showed that all exosome groups could activate macrophages,including promoting cell proliferation and increasing the secretion of NO,IL-1β,TNF-α and IL-6.Among them,exosomes in fresh milk and breast milk showed obvious immune-activating effects.The effect of fresh milk group on IL-1β was significantly higher than that of milk products group(P<0.05);there was no significant difference between the lyophilized powder group and the freeze-dried powder group in promoting TNF-α and IL-6,which was better than the UHT milk group and formula powder group.4.Study on inhibition of LPS-mediated inflammation by exosomes from different milk productsLPS-induced macrophage inflammation model was established to explore the effects of different milk products exosomes on the inhibition of inflammatory response.The results showed that the breast milk exosome group had the best effect on inhibiting LPS-induced ROS accumulation and pro-inflammatory factor level increase in macrophages.Exosomes in fresh milk group inhibited the production of NO and IL-6,and down-regulated the expression of IL-1βm RNA significantly higher than those in milk products group.Fresh milk group and freeze-dried powder group had better ability to reduce IL-6 and TNF-α transcription levels than UHT milk group and formula powder group.The ROS production of fresh milk group was not significantly different from that of freeze-dried powder group and UHT milk group,but was significantly lower than that of formula powder group(P<0.05).In addition,exosomes in each group can decrease the expression of TLR4/NF-κB signaling pathway induced by LPS to different degrees.The protein expression of TLR4 receptor in breast milk,fresh milk,freeze-dried powder group and UHT milk group had no statistical difference,which was significantly higher than that in formula powder group.The inhibitory effect of fresh milk group on the phosphorylation of p65 and IκBα was significantly lower than that of breast milk group.The protein expression levels of p-p65 and p-IκBα in fresh milk group were similar to those in freeze-dried powder group,and significantly higher than those in UHT milk group.The inhibition of phosphorylation of p65 and IκBα protein in formula group was the worst.These results suggest that exosomes extracted from breast milk,fresh cow milk,pasteurized milk,freeze-dried powder and formula powder all have a bidirectionally regulated effect on the immune system,namely,up-regulating the immune activity of normal macrophages and down-regulating the inflammatory response of macrophages induced by LPS.The immunoregulatory effect of fresh milk group was similar to that of freeze-dried powder group,and higher than that of UHT milk group.The immunoregulatory effect of formula powder group was the lowest.This may be related to different levels of exosomes regulating the expression of immune-related mi RNAs in macrophages in each group.更多还原