粪菌移植对溃疡性结肠炎小鼠结肠黏膜Th1/Th2细胞及其细胞因子表达的影响

Effects of fecal microbiota transplantation on expression of Th1/Th2 cells and related cytokines in DSS-induced colitis mice

目的观察粪菌移植(FMT)对结肠炎小鼠结肠组织内Th1/Th2平衡及其细胞因子的影响。方法用葡聚糖硫酸钠(DSS)制备溃疡性结肠炎小鼠模型,共48只雄性C57BL/6小鼠,按3∶2∶1随机分为干预组、对照组和粪便供体组。其中干预组24只小鼠自由饮用2%DSS溶液,第6天时再次称重随机分为3组,每组6只:粪便滤液组(A组)、美沙拉嗪缓释颗粒组(B组)、生理盐水组(C组),并分别灌肠给予粪便滤液(500mg/mL,0.3mL/次)、美沙拉嗪缓释颗粒混悬液(25mg/mL,0.3mL/次)和生理盐水(0.3mL/次),每日2次,连续5d;对照组分为急性结肠炎组(D组)和正常对照组(E组),每组6只,分别自由饮用2%DSS溶液和蒸馏水7d后处死;粪便供体组(F组)正常进食饮水,采集新鲜粪便制作粪便滤液。评估疾病活动指数、脾脏指数、结肠长度和结肠组织病理评分。免疫组化法和酶联免疫吸附试验(ELISA)分别检测结肠组织Th1/Th2细胞及细胞因子(TNF-α、IFN-γ、IL-4和IL-10)的表达。结果 (1)粪菌移植治疗明显改善结肠挛缩和病理损伤(P<0.05),而脾脏指数明显增加(P<0.05);(2)粪菌移植使结肠炎小鼠结肠组织的T-bet阳性细胞和Gata-3阳性细胞表达升高,T-bet/Gata-3平衡右移;(3)粪菌移植使结肠炎小鼠结肠组织细胞因子(TNF-α、IFN-γ、IL-4、IL-10)水平下降。结论粪菌移植促进结肠炎小鼠的结肠黏膜修复,可能与调节结肠组织Th1/Th2细胞平衡,下调炎症因子,介导Th2免疫修复有关。

Objective To observe the effect of fecal microbiota transplantation（FMT）on the balance of mucosal Th1/Th2 cells,and related cytokine levels in colitis induced by dextran sulfate sodium（DSS）.Methods Forty-eight male C57 BL/6 mice were randomly divided into intervention group,control group or feces donor group at a ratio of 3∶2∶1.The twenty-four mice in the intervention group were given 2% DSS solution for 5 days,which were subdivided into FMT（Group A）,etiasa（Group B）or saline（Group C）group randomly on the 6 th day,and received rectal administration of fecal filtrate（500 mg/mL,0.3 mL/time）,etiasa suspension（25 mg/mL,0.3 mL/time）or saline（0.3 mL/time）respectively,b.i.d.for five consecutive days.The mice in control group were divided into acute colitis（Group D）or normal control（Group E）group,drinking 2% DSS solution or distilled water respectively for 7 days,and were then executed on the 8 th day.The fresh faeces from mice donors（Group F）were collected for preparation of fecal filtrate.The disease activity index（DAI）,spleen index（SI）,length and histopathological index of colon were evaluated.The expression of Gata-3/T-bet positive cells and cytokines such as TNF-α,IFN-γ,IL-4 and IL-10 were determined with immunohistochemical method and enzyme-linked immunosorbent assay（ELISA）respectively.Results（1）FMT significantly improved the DAI,length and pathological index of colon in mice with colitis（P0.05）,while the SI increased obviously（P0.05）.（2）FMT increased T-bet and Gata-3 positive cell infiltration in colon tissues and the balance of T-bet/Gata-3 moved towards the right in mice with DSS induced colitis.（3）FMT significantly reduced the expressions of TNF-α,IFN-γ,IL-4 and IL-10 in the colon tissue in DSS-induced colitis mice.Conclusion FMT can regulate the balance of Th1/Th2 cells in colonic tissue,reduce the levels of IL-4,IL-10,TNF-αand IFN-γ,and improve the colonic histological index in C57 BL/6 mice with DSS-induced colitis.