粪菌移植对非肥胖糖尿病小鼠发生1型糖尿病的影响及其机制探讨  

Effect of fecal microbiota transplantation on type 1 diabetes mellitus in non-obese diabetic mice and its underlying mechanism

作　　者：周迎 李阳阳 刘煜 Zhou Ying;Li Yangyang;Liu Yu(Department of Endocrinology,Sir Run Run Hospital Affiliated to Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学附属逸夫医院内分泌科,南京211166

出　　处：《中华医学杂志》2022年第16期1224-1231,共8页National Medical Journal of China

基　　金：国家自然科学基金(82070849,81770778,81800735);江苏省双创团队项目(20182015)。

摘　　要：目的:探讨粪菌移植(FMT)对非肥胖糖尿病(NOD)小鼠发生1型糖尿病(T1DM)的影响及相关机制。方法:8~9周龄的雌性NOD小鼠72只,随机数字表法分为对照组(n=36)和FMT组(n=36)。FMT组移植C57BL/6鼠的粪菌,对照组移植自身粪菌,隔日1次,共5次。比较两组小鼠的胰岛炎评分和T1DM发病率;通过16S rRNA基因测序评估NOD小鼠粪菌组成;实时定量PCR检测肠屏障相关基因的表达水平;流式细胞术检测肠-胰腺免疫轴中调节性T细胞(Treg)、辅助性T细胞(Th)-1及Th17的比例;通过氨基酸代谢组学检测血清中氨基酸的水平。结果:26周龄时FMT组的T1DM发病率为40.9%(9/22),低于对照组的72.7%(13/22)(P=0.034)。FMT促进了肠道中有益菌,如Lactobacillus、Clostridium_sp_ND2、Candidatus_Arthromitus和Clostridiaceae_1的定植。与对照组相比,FMT组肠屏障相关基因的mRNA表达上调[黏蛋白(Muc)-1:0.93±0.29比2.97±0.79,P=0.036;Muc2:0.72±0.39比10.70±3.54,P=0.019;Muc3:1.79±0.69比10.97±2.78,P=0.009;Muc4:1.01±0.23比2.42±0.49,P=0.029;封闭蛋白(Ocln):0.96±0.08比1.81±0.36,P=0.045;闭合蛋白(Cldn)-1∶0.94±0.17比2.20±0.43,P=0.022]。与对照组相比,FMT组肠系膜淋巴结、胰腺淋巴结、派伊尔结中Treg比例增加[(6.10±0.49)%比(7.54±0.27)%,P=0.020;(5.28±0.39)%比(6.42±0.34)%,P=0.048;(6.78±0.42)%比(7.88±0.13)%,P=0.029];Th1比例减少[(1.02±0.06)%比(0.83±0.06)%,P=0.040;(0.82±0.10)%比(0.56±0.05)%,P=0.038;(1.28±0.12)比(0.85±0.07),P=0.012],Th17比例减少[(0.40±0.01)%比(0.30±0.02)%,P=0.004;(0.40±0.02)%比(0.31±0.02)%,P=0.008;(0.51±0.06)比(0.36±0.02),P=0.027)]。与对照组相比,FMT组小鼠血清中亮氨酸[(92.86±7.32)比(91.87±12.62)μmol/L,P=0.027)]、缬氨酸[(162.74±15.97)比(155.89±25.70)μmol/L,P=0.046)]、异亮氨酸[(75.65±5.59)比(73.61±9.67)μmol/L,P=0.048)]含量减少。结论:FMT可减轻NOD小鼠的胰岛炎并减缓T1DM的发生,其机制可能与重塑NOD小鼠肠道菌群并改善肠屏障功能、影响肠-胰腺�Objective To investigate the effects of fecal microbiota transplantation(FMT)on non-obese diabetic(NOD)mice of type 1 diabetes mellitus(T1DM)and its underlying mechanisms.Methods A total of 8-9 week-female NOD mice were randomly divided into control(n=36)and FMT groups(n=36)according to the random number table.Fecal microbiota from C57BL/6 mice were transplanted into FMT group,and control group were transplanted with microbiota from themselves,once every two days for 5 times.The insulitis score and incidence of T1DM were compared between two groups;16S rRNA gene sequencing was used to evaluate the structure of fecal bacteria in NOD mice.The expressions of intestinal barrier related genes were detected by real-time quantitative PCR.The proportions of regulatory T cells(Tregs),helper T cell(Th)-1 and Th17 in the enteric-pancreatic immune axis were detected by flow cytometry.Amino acid in serum was measured by amino acid metabolomics.Results Incidence of T1DM in NOD mice from FMT group was 40.9%(9/22),lower than 72.7%(13/22)from control group at 26 weeks of age(P=0.034).FMT promoted colonization of probiotics such as Lactobacillus,Clostridium_sp_ND2,Candidatus_Arthromitus and Clostridiaceae_1;mRNA of intestinal barrier related genes were up-regulated in FMT group[mucins(Muc)-1:0.93±0.29 vs 2.97±0.79,P=0.036;Muc2:0.72±0.39 vs 10.70±3.54,P=0.019;Muc3:1.79±0.69 vs 10.97±2.78,P=0.009;Muc4:1.01±0.23 vs 2.42±0.49,P=0.029;Occludin(Ocln):0.96±0.08 vs 1.81±0.36,P=0.045;Claudin(Cldn)-1:0.94±0.17 vs 2.20±0.43,P=0.022]compared to control.The proportions of Treg in mesenteric lymphoid node,pancreatic lymph node and peyer′s patches of FMT group[(6.10±0.49)%vs(7.54±0.27)%,P=0.020;(5.28±0.39)%vs(6.42±0.34)%,P=0.048;(6.78±0.42)%vs(7.88±0.13)%,P=0.029]were increased compared to control,while proportions of Th1[(1.02±0.06)%vs(0.83±0.06)%,P=0.040;(0.82±0.10)%vs(0.56±0.05)%,P=0.038;(1.28±0.12)vs(0.85±0.07),P=0.012]and proportions of Th17[(0.40±0.01)%vs(0.30±0.02)%,P=0.004;(0.40±0.02)%vs(0.31±0.02)%,P=0.008;

关 键 词：糖尿病 1型 粪菌移植 肠道菌群 NOD小鼠 实验研究 

分 类 号：R587.1[医药卫生—内分泌]