牛乳来源外泌体对巨噬细胞极化调控作用的研究

Study on the Regulation of Macrophage Polarization by Exosomes Derived from Cow Milk

本研究旨在探究不同牛乳来源外泌体(exosomes,Exo)对巨噬细胞极化的影响。无菌采集10份牛乳样品,通过体细胞数(somatic cell count,SCC)测定和细菌学鉴定判断乳样性质;差速超速离心法富集纯化牛乳来源Exo;通过纳米颗粒跟踪分析(NTA)、透射电镜(TEM)、Western blot(WB)鉴定Exo;荧光示踪法检测Exo能否被巨噬细胞(Raw264.7)内化;实时荧光定量PCR(RT-PCR)、酶联免疫吸附试验(ELISA)检测牛乳来源Exo作用于Raw264.7细胞后促炎性细胞因子、抗炎性细胞因子mRNA水平及蛋白水平的变化。通过SCC判定有4份正常乳样,6份炎症乳样;从炎症乳样中分离出4株致病菌,生物学特性分别与大肠杆菌、绿脓杆菌和克雷伯肺炎杆菌基本一致,经16S rDNA基因测序比对分析证实菌株为上述致病菌;超速离心法分离得到10份乳样Exo;不同牛乳样本来源Exo与Raw264.7共培养6 h后均能被其内化;与对照(Con)组相比,低体细胞数来源Exo(L-Exo)组和高体细胞数来源Exo(H-Exo)组均能显著增加Raw264.7细胞肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6的mRNA和蛋白表达水平(P<0.05),一氧化氮合酶(iNOS)和巨噬细胞甘露糖受体CD206蛋白表达显著升高(P<0.05),IL-10、转化生长因子(TGF)-β的mRNA和蛋白水平下降(P<0.05),精氨酸酶(Arg)-1 mRNA表达水平下降(P<0.05)。在奶牛乳腺炎症反应过程中,炎症奶牛牛乳来源Exo可促进巨噬细胞向M1型极化,高表达促炎性细胞因子,促进炎症反应,而乳腺健康状态下分泌的Exo能介导巨噬细胞向M2型极化,表达高水平的抗炎性细胞因子,参与机体免疫调节,有一定的抗炎作用,证明牛乳Exo可调节巨噬细胞极化过程。

The aim of this study was to investigate the effects of exosomes(Exo)from different milk sources on macrophage polarization.Ten milk samples were collected aseptically and the properties of the milk samples were determined by somatic cell count(SCC)and bacteriological identification;the bovine milk-derived exosomes were enriched and purified by differential ultracentrifugation;exosomes were identified by nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM)and western blot(WB);the fluorescent tracer method was used to detect whether exosomes could be internalized by macrophages(Raw264.7);real-time fluorescent quantitative PCR(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to detect changes in mRNA levels of pro-inflammatory cytokines,anti-inflammatory cytokines and protein levels after the action of bovine milk-derived exosomes on Raw264.7 cells.According to SCC,there were 4 normal milk samples and 6 inflammatory milk samples.Four strains of pathogenic bacteria were isolated from milk samples of inflamed cows and their biological characteristics were consistent with Escherichia coli,Pseudomonas aeruginosa and Klebsiella pneumoniae respectively;the exosomes were separated by ultracentrifugation and different bovine milk-derived exosomes were internalized by Raw264.7 after co-culture with it for 6 h.Compared with the control group(Con),compared with the control(Con)group,both the low somatic cell count source exosomes(L-Exo)and high somatic cell count source exosomes(H-Exo)groups significantly increased Raw264.7 cell tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6 mRNA and protein expression levels(P<0.05),nitric oxide synthase(iNOS)and macrophage mannose receptor CD206 protein expression were significantly increased(P<0.05),IL-10,transforming growth factor(TGF)-βmRNA and protein levels were decreased(P<0.05),arginase(Arg)-1 mRNA expression levels were decreased(P<0.05).During the inflammatory response in the mammary gland of cows,mastitis bovine milk-derived exosomes can promote the polarisation of macrophages towards M1 type,express high levels of pro-inflammatory cytokines and promote the inflammatory response,while exosomes secreted in the healthy state of the mammary gland can mediate the polarisation of macrophages towards M2 type,express high levels of anti-inflammatory cytokines,participate in the immune regulation of the body and have certain anti-inflammatory effects,this demonstrates that milk-derived exosomes can influence the process of macrophage polarization.