生命早期补充鼠李糖乳杆菌GG保护子代肠道屏障的研究

摘要

目的 探讨生命早期益生菌鼠李糖乳杆菌GG (LGG)肠道定植对子代小鼠肠道屏障及肠道发育的影响及其可能的作用机制.方法 选择SPF级6只6周龄野生型C57BL/6雌鼠随机分为实验组和对照组,实验组为LGG活菌组,对照组为LGG灭活菌组,分别予108 cfu/ml LGG活菌或LGG灭活菌灌胃直至自然分娩.两组子代小鼠分别于出生第1～5天继续给予107 cfu/ml LGG活菌或LGG灭活菌灌胃.记录3周子代小鼠体重变化;于第2、3周检测子代小鼠LGG菌的定植情况;运用Real-time PCR方法评价肠道促炎因子及紧密连接分子mRNA变化;运用HE、免疫组化、免疫荧光染色法及酶联免疫吸附法评价3周子鼠的肠道屏障情况.结果 实验组的子代小鼠与对照组相比,第1周时体重相比无明显差异,第2周和第3周体重有所增加[第2周:(4.326±0.140)g比(3.790±0.240)g,t=3.707,P=0.006;第3周:(8.040±0.370)g比(7.295±0.326)g,t=3.130,P=0.011];仅在实验组子代小鼠的粪便中可检测出LGG定植.肠道定植可促进小肠绒毛长度及结肠隐窝深度明显增长[空肠:(320.000±22.514) μm比(265.100±15.611) μm,t=8.258,P＜0.001;回肠(150.500±13.099) μm比(111.000±11.308) μm,t=9.958,P＜0.001;结肠(295.000±15.209) μm比(233.100±6.678) μm,t=9.129,P＜0.001].与LGG灭活组相比,实验组子代小鼠结肠隐窝中杯状细胞的数量增加[(35.24±1.370)个比(11.62±0.780)个,t=15.000,P＜0.001],促炎因子IFN-γ(0.512±0.206比1.280±0.232,t=4.970,P=0.001)、IL-6(0.941±0.215比1.364±0.271,t=2.452,P=0.040)、IL-10 (0.744±0.294比1.341±0.320,t=2.762,P=0.025)和TNF-α(2.581±0.500比3.702±0.150,t=2.553,P=0.034)mRNA的相对表达水平降低,肠道组织紧密连接分子(Claudin3)(1.881±0.172比1.283±0.152,t=4.932,P=0.001)和闭锁蛋白分子(Occludin)(1.164±0.342比0.812±0.224,t=3.67,P=0.016)表达含量显著增加(P＜0.01).结论 生命早期LGG定植可通过抑制肠道低度炎症进而保护肠道屏障.本研究将为生命早期补充益生菌进而防治肠道疾病奠定实验基础.%Objective To investigate the effects of Lactobacillus rhamnosus GG (LGG) colonization in early life on intestinal barrier and intestinal development in offspring mice and its possible mechanism.Methods Six C57BL/6 pregnant mice with the same conception time of 6 weeks were selected and randomly divided into experiment group given 108 cfu/ml LGG live bacteria and control group given LGG inactivated bacteria by gavage from the 18th day of pregnancy until natural birth.The progeny mice in the two groups were continued to be gavaged with 107 cfu/ml of LGG live bacteria or LGG inactivated bacteria on days 1-5 of birth.The body weight changes of 3 week'progeny mice were recorded.The colonization of LGG bacteria in offspring mice was detected at 2nd and 3rd weeks.The mRNA of intestinal proinflammatory cytokines and tight junction molecules were evaluated by real-time PCR method.HE,immunohistochemistry,immunofluorescence staining and enzyme-linked immunosorbent assay were used to evaluate the intestinal barrier of 3-week old off spring mice.Results Compared with the control group,the progeny mice of the experiment group showed no significant difference in body weight at the first week,and the body weight increased at the second week and the third week [2ndweek:(3.790±0.240) g vs.(4.326±0.140) g,t=3.707,P=0.006;3rd week:(7.295±0.326) g vs.(8.040±0.370) g,t=3.130,P=0.011].LGG colonization can be detected only in the feces of progeny mice in the experiment group.Intestinal colonization can promote the growth of small intestine villi and colon crypt depth [jejunum:(320.000±22.514) μm vs.(265.100±15.611) μm,t=8.258,P＜0.001;ileum:(150.500±13.099) μm vs.(111.000±11.308) μm,t=9.958,P＜0.001;colon:(295.000±15.209) μm vs.(233.100±6.678) μm,t=9.129,P＜0.001].Compared with the control group,the number of goblet cells in the colonic crypt of the experiment group increased (11.62 ± 0.780 vs.35.24 ±1.370,t=15.000,P＜0.001),and the relative mRNA expression levels of pro-inflammatory factors as IFN-γ (1.280±0.232 vs.0.512±0.206,t=4.970,P=0.001),IL-6 (1.364±0.271 vs.0.941±0.215,t=2.452,P=0.040),IL-10 (1.341±0.320vs.0.744±0.294,t=2.762,P=0.025)andTNF-α (3.702±0.150 vs.2.581±0.500,t=2.553,P=0.034) in the experiment group decreased;the expression levels of the intimate tight junction molecules (Claudin3) (1.283±0.152 vs.1.881±0.172,t=4.932,P=0.001) and the atresia protein molecule (Occludin) (1.164±0.342 vs.0.812±0.224,t=3.67,P=0.016) significantly increased.Conclusion Early life LGG colonization protects the intestinal barrier by inhibiting lowgrade intestinal inflammation.This study will lay the experimental foundation for the supplementation of probiotics in early life so as to prevent intestinal diseases.