粪菌移植对坏死性小肠结肠炎新生小鼠肠道菌Th1/Th2细胞因子表达的影响

Effect of fecal bacteria transplantation on intestinal flora and expression of Th1/Th2 cytokines in neonatal mice with necrotizing enterocolitis

目的:探讨粪菌移植(FMT)对新生儿坏死性小肠结肠炎(NEC)小鼠肠道菌群及Th1/Th2细胞因子表达的影响。方法:将48只3日龄C57BL/6新生小鼠随机分为4组:对照组、NEC模型组、FMT预防组、FMT治疗组,每组12只。除对照组外,其余三组均采用缺氧+冷刺激+人工喂养方法连续刺激3 d建立NEC模型。对照组幼鼠自由饮水6 d;FMT预防组在建立NEC模型同时灌胃给予FMT,自由饮水恢复3 d。FMT治疗组在模型建成后连续3 d灌胃给予FMT,随后隔夜空腹处死各组新生小鼠。记录各组小鼠实验期间体重变化和生存情况,收集每日粪便进行肠道菌群分析,肉眼及HE染色观察回盲部肠组织病理变化,免疫组化法和ELISA分别检测结肠组织Th1/Th2细胞及其细胞因子(IL-4、IL-10、TNF-α及IFN-γ)表达。结果:FMT预防组及FMT治疗组小鼠体质量及生存率明显高于NEC模型组(P<0.05)。与NEC模型组相比,FMT预防组及FMT治疗组小鼠结肠炎症均明显缓解,隐窝数增加,结肠黏膜呈愈合趋势,T-bet阳性细胞减少(P<0.05),Gata-3阳性细胞增加(P<0.05),T-bet/Gata-3右移,Th2细胞因子(IL-4、IL-10)表达明显增加(P<0.05),Th1细胞因子(TNF-α、IFN-γ)表达明显降低(P<0.05),且FMT预防组及FMT治疗组均能明显促进NEC小鼠乳杆菌属增殖而抑制拟杆菌属和颤螺菌属生长。结论:FMT预防组和治疗组均可促进NEC模型小鼠乳杆菌属增殖,抑制拟杆菌属和颤螺菌属生长而减轻结肠炎症损伤,促进肠黏膜屏障功能恢复,可能与调节Th1/Th2平衡,介导Th2免疫修复有关。

Objective:To investigate effects of fecal microbiota transplantation(FMT)on intestinal flora and Th1/Th2 cytokine expression in neonatal necrotizing enterocolitis(NEC)mice.Methods:48 3 day-old C57 BL/6 newborn mice were divided into four groups randomly:control group,NEC model group,FMT prevention group and FMT treatment group,with 12 mice in each group. Except for control group,the other three groups were stimulated by method of hypoxia+cold stimulation+artificial feeding for 3 d consecutively to establish NEC model. Control group was free to drink pure water for 6 d;FMT prevention group was given FMT gavage treatment while establishing NEC model,and restored by purified water for 3 d;FMT treatment group was given FMT gavage treatment for 3 d consecutively after model was established,and then overnight fasting each group of newborn mice were sacrificed. Body mass changes and survival were recorded during experiment in each group. Daily feces were collected for intestinal flora analysis,and ileocecal intestinal pathological changes were observed by naked eye and HE staining. Immunohistochemistry and ELISA were used to detect expressions of Th1/Th2 cells and their cytokines(IL-4,IL-10,TNF-α and IFN-γ)in colon tissue.Results:Body weight of mice in FMT prevention group and FMT treatment group was higher than NEC model group. Compared with NEC model group,colonic inflammation in FMT prevention group and FMT treatment group was significantly relieved,number of crypts was increased,colonic mucosa showed a healing trend,T-bet positive cells number was decreased(P<0.05),and Gata-3 positive cells number was increased(P<0.05),T-bet/Gata-3 shifted to right,Th1 cytokines(TNF-α,IFN-γ)expressions were significantly decreased(P<0.05),and Th2 cytokines(IL-4,IL-10)expressions were significantly increased(P<0.05),and both groups significantly promoted proliferation of Lactobacillus in NEC mice and inhibit growth of Bacteroides and Treponema.Conclusion:Both FMT prevention group and treatment group can promote proliferation of Lactobacillus in NEC model,inhibit growth of Bacteroides and Treponema,reduce inflammation of colonic inflammation,promote recovery of intestinal mucosal barrier function,which possibly related to regulating balance of Th1/Th2 and mediating Th2 immune repair.