利用2'-岩藻糖基乳糖生长的两歧双歧杆菌BD-1免疫调节功能评价  

Evaluation of the immunomodulatory function of Bifidobacterium bifidum BD-1 grown with 2'-fucosyllactose

作　　者：许晓林 王斯栌 陈菲 逯畅 李倪亚 程如越 何方 沈曦 XU Xiaolin;WANG Silu;CHEN Fei;LU Chang;LI Niya;CHENG Ruyue;HE Fang;SHEN Xi(West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]四川大学华西公共卫生学院/华西第四医院,四川成都610041

出　　处：《中国微生态学杂志》2024年第7期745-752,760,共9页Chinese Journal of Microecology

基　　金：国家自然科学基金面上基金项目(81973042)。

摘　　要：目的探究两歧双歧杆菌BD-1(Bifidobacterium bifidum BD-1,BD-1)以2-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)作为唯一碳源时,与葡萄糖相比,其菌体和代谢产物诱导小鼠巨噬细胞系RAW264.7细胞分泌细胞因子的效应,以探索BD-1菌利用母乳低聚糖后免疫调节的菌株特性。方法(1)菌体部分干预实验:将BD-1活菌接种于不同碳源的改良MRS肉汤培养基中,改良过的MRS肉汤培养基包括以2%2'-FL作为唯一碳源(2'-FL组)和以2%葡萄糖作为唯一碳源(Glu组)的培养基。将接种后的BD-1活菌在厌氧条件下培养24 h后,分离各组菌体和培养基上清液。将菌体部分与RAW264.7细胞共同培养,并以肽聚糖(peptidoglycan,PGN)为菌体成分对照,同时设置不添加干预物的阴性对照(Ctrl组)。(2)菌体代谢产物部分干预实验:将培养基上清液与RAW264.7细胞共同培养,以含有2%2'-FL或葡萄糖但未进行BD-1菌发酵处理的培养基作为实验对照,并设置不添加干预物的培养基与RAW264.7细胞的共培养上清液作为阴性对照(Ctrl组),以PGN与RAW264.7细胞的共培养上清液作为阳性对照(PGN组)。培养24 h后,提取RAW264.7细胞总RNA,逆转录后采用实时荧光定量PCR检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6、IL-10、IL-12、IL-1β的基因表达量;收集各组受试物与RAW264.7细胞的共培养上清液,采用酶联免疫吸附试验检测共培养上清液中上述细胞因子的分泌量。结果(1)菌体部分干预:与PGN组相比,菌体部分2'-FL组和Glu组的TNF-α、IL-6、IL-10分泌量及基因表达量显著上升(均P<0.05)。与Glu组相比,2'-FL组的IL-6、IL-10分泌量显著下降(均P<0.05),IL-6、IL-10的基因表达具有下降趋势。(2)菌体代谢产物部分干预:BD-1-2'-FL上清组和BD-1-Glu上清组的TNF-α、IL-6及IL-10分泌水平以及基因表达水平较2'-FL组和Glu组(未进行BD-1菌发酵处理的组别)有显著提高(均P<0.05),而且2'-FL组中IL-6Objective To explore the effects of Bifidobacterium bifidum BD-1(BD-1)and its metabolites in inducing cytokine secretion in murine macrophage cell line RAW264.7 cells when using 2'-fucosyllactose(2'-FL)vs glucose as the sole carbon source,aiming to gain further insights into the immunomodulatory properties of BD-1 when using human milk oligosaccharides as a carbon source.Methods(1)BD-1 intervention experiment:live BD-1 bacteria were inoculated in modified MRS broth containing 2%2'-FL(2'-FL group)and 2%glucose as the sole carbon source(Glu group)respectively.Following anaerobic incubation for 24 hours,the viable BD-1 bacteria were separated from the cells and medium supernatants in each group.The cell components were co-cultured with RAW264.7 cells,with peptidoglycan(PGN)as the control for cellular composition.A negative control group without any intervention(Ctrl group)was established.(2)Intervention experiments with metabolites of BD-1:the experimental control consisted of medium containing 2%2'-FL or glucose without BD-1 fermentation,while the negative control(Ctrl group)involved medium without any intervention with RAW264.7 cells.The co-culture supernatant of PGN and RAW264.7 cells was utilized as a positive control(PGN group).Following 24 hours of incubation,the total RNA from RAW264.7 cells was extracted and subjected to reverse transcription using quantitative real-time PCR.The mRNA expression levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-10,IL-12 and Il-1βwere assessed through RT-qPCR.Furthermore,the supernatants obtained from the co-cultures of RAW264.7 cells and the respective test materials in each group were collected to determine the secretion levels of these cytokines via enzyme-linked immunosorbent assay.Results(1)BD-1 intervention:compared to the PGN group,the secretion and gene expression of TNF-α,IL-6,and IL-10 were significantly elevated in both the 2'-FL group and Glu group(all P<0.05).In comparison to the Glu group,there was a significant decrease in the secretion of IL-6 and

关 键 词：两歧双歧杆菌 母乳低聚糖 RAW264.7细胞 肿瘤坏死因子-Α 白细胞介素 

分 类 号：R151[医药卫生—营养与食品卫生学]