基于人消化道微生态体外模拟系统观察纳米二氧化钛对肠道菌群的影响  

Effects of nano titanium dioxide on gut microbiota based on human digestive tract microecology simulation system in vitro

作　　者：张家赫 史佳琪 陈章健[1] 贾光[1] ZHANG Jia-he;SHI Jia-qi;CHEN Zhang-jian;JIA Guang(Department of Occupational and Environmental Health Sciences,Peking University School of Public Health,Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety,Beijing 100191,China)

机构地区：[1]北京大学公共卫生学院劳动卫生与环境卫生学系,食品安全毒理学研究与评价北京市重点实验室,北京100191

出　　处：《北京大学学报（医学版）》2022年第3期468-476,共9页Journal of Peking University:Health Sciences

基　　金：国家重点研发计划重点专项(2017YFC1600200);国家自然科学基金(81703257)。

摘　　要：目的:通过消化道微生态体外模拟系统探索纳米二氧化钛(titanium dioxide nanoparticles,TiO_(2)NPs)对人源肠道菌群组成和结构的影响。方法:对TiO_(2)NPs进行粒径、形状、晶型和团聚程度的表征。通过模拟胃、小肠、结肠的液体环境和物理条件建立体外人消化道微生态模拟系统,并对模拟系统的稳定性进行评价。从人粪便中提取菌群,采用该模拟系统稳定培养,分别暴露于0、20、100、500 mg/L TiO_(2)NPs中,收集染毒24 h后的菌液,通过16S rRNA测序技术分析TiO_(2)NPs对人源肠道菌群组成和结构的影响,利用线性判别效应量分析(linear discriminant analysis effect size,LEfSe)筛选差异细菌,根据京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)数据库进行功能预测。结果:TiO_(2)NPs为球形粒径,直径(25.12±5.64)nm,晶体结构为锐钛矿,在超纯水中水合粒径为(609.43±60.35)nm,Zeta电位为(-8.33±0.22)mV。体外消化道模拟系统培养人源肠道菌群24 h后达到相对稳定状态,肠球菌(Enterococci)、大肠杆菌(Enterobacterium)和乳酸杆菌(Lactobacillus)计数可分别达到(1.60±0.85)×10^(7)个、(5.60±0.82)×10^(7)个和(2.70±1.32)×10^(7)个。16S rRNA测序结果显示,与对照组相比,TiO_(2)NPs染毒组(20、100和500 mg/L)在门、纲、目、科、属水平上,肠道菌群的物种数量和均匀度未受到明显影响,但部分菌种的相对丰度发生显著变化。TiO_(2)NPs染毒组(20、100和500 mg/L)与对照组之间共筛选出42种不同的差异细菌(线性判别分析分数,linear discriminant analysis score,LDA score>3),以肠杆菌属(Enterobacter)、拟杆菌科(Bacteroidaceae)、乳酸杆菌科(Lactobacillaceae)、双歧杆菌科(Bifidobacteriaceae)和梭菌属(Clostridium)等为代表。进一步的肠道菌群功能预测分析显示,TiO_(2)NPs可能影响肠道菌群的氧化磷酸化、能量代谢、磷酸盐和磷酸盐代谢、甲烷代谢等代谢和功能(P<0.05)。结论Objective:To explore the effects of oral exposure to titanium dioxide nanoparticles(TiO_(2) NPs)on the composition and structure of human gut microbiota.Methods:The particle size,shape,crystal shape and degree of agglomeration in ultrapure water of TiO_(2) NPs were characterized.The in vitro human digestive tract microecological simulation system was established by simulating the fluid environment and physical conditions of stomach,small intestine and colon,and the stability of the simulation system was evaluated.The bacterial communities were extracted from human feces and cultured stably in the simulated system.They were exposed to 0,20,100 and 500 mg/L TiO_(2) NPs,respectively,and the bacterial fluids were collected after 24 h of exposure.The effect of TiO_(2) NPs on the composition and structure of human gut microbiota was analyzed by 16S rRNA sequencing technology.Linear discriminant analysis effect size(LEfSe)was used to screen differential bacteria,and the Kyoto encyclopedia of genes and genomes(KEGG)database for functional prediction.Results:The spherical and anatase TiO_(2) NPs were(25.12±5.64)nm in particle size,while in ultra-pure water hydrated particle size was(609.43±60.35)nm and Zeta potential was(-8.33±0.22)mV.The in vitro digestive tract microecology simulation system reached a relatively stable state after 24 hours,and the counts of Enterococci,Enterobacte-rium,and Lactobacillus reached(1.6±0.85)×10^(7),(5.6±0.82)×10^(7) and(2.7±1.32)×10^(7),respectively.16S rRNA sequencing results showed that compared with the control group,the number and evenness of gut microbiota were not significantly affected at phylum,class,order,family and genus levels in TiO_(2) NPs groups(20,100 and 500 mg/L).The relative abundance of some species was significantly changed,and a total of 42 different bacteria were screened between the TiO_(2) NPs groups(20,100 and 500 mg/L)and the control group[linear discriminant analysis(LDA)score>3],represented by Enterobacter,Bacteroidaceae,Lactobacillaceae,Bifidobacteriace

关 键 词：纳米粒子 二氧化钛 胃肠道微生物组 模型 生物学 16S rRNA测序技术 

分 类 号：R155.5[医药卫生—营养与食品卫生学]