粪菌移植干预治疗溃疡性结肠炎的免疫学机制研究

doi:10.12114/j.issn.1007-9572.2021.02.069

中国全科医学 ›› 2022, Vol. 25 ›› Issue (03): 298-304.DOI: 10.12114/j.issn.1007-9572.2021.02.069

粪菌移植干预治疗溃疡性结肠炎的免疫学机制研究

翁剑锋¹, 徐佳², 刘朋¹, 葛巍¹, 陈教华¹, 彭迎迎³, 胡家丽⁴, 崔曼曼⁴, 张磊昌^1,^*

1.330006　江西省南昌市，江西中医药大学附属医院
2.510405　广东省广州市，广州中医药大学第一附属医院
3.330012　江西省南昌市，江西中医药大学第二附属医院
4.330004　江西省南昌市，江西中医药大学研究生院

收稿日期:2021-04-28 修回日期:2021-09-01 出版日期:2022-01-20 发布日期:2021-12-29
通讯作者: 张磊昌
基金资助:
国家自然科学基金资助项目(81804101)

Immunological Mechanism of Faecal Bacterial Transplantation in the Intervention of Ulcerative Colitis

WENG Jianfeng1，XU Jia2，LIU Peng1，GE Wei1，CHEN Jiaohua1，PENG Yingying3，HU Jiali4，CUI Manman4，ZHANG Leichang1*

1.Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine，Nanchang 330006，China
2.The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine，Guangzhou 510405，China
3.The Second Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine，Nanchang 330012，China
4.Graduate School of Jiangxi University of Traditional Chinese Medicine，Nanchang 330004，China
*Corresponding author：ZHANG Leichang，Deputy chief Chinese physician；E-mail：acme322@163.com

Received:2021-04-28 Revised:2021-09-01 Published:2022-01-20 Online:2021-12-29

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摘要/Abstract

摘要： 背景溃疡性结肠炎被世界卫生组织列为现代难治疾病之一。目前西医对其治疗尚存在诸多不足之处，研究显示粪菌移植（FMT）对其有一定的疗效，但机制尚不明确。目的采用FMT治疗小鼠溃疡性结肠炎模型，验证FMT的疗效和可能的作用机制。方法2019年12月至2020年4月，采用随机数字表法将60只小鼠分为正常对照组（Control组）、溃疡性结肠炎模型组（Model组）、溃疡性结肠炎模型+粪菌移植治疗组（Model+FMT组）和溃疡性结肠炎模型+5-氨基水杨酸（5-ASA）治疗组（Model+5-ASA组），各15只。Control组不作任何干预处理；Model组制备小鼠溃疡性结肠炎模型；Model+FMT组于造模成功后，给予制备的粪菌液0.2 ml/次灌肠；Model+5-ASA组于造模成功后，给予0.019 5 g/ml 5-ASA灌肠。通过透射电镜观察肠组织超微结构变化，流式细胞检测血液辅助性T细胞（Th）-17、Th-1、Th-2、Treg细胞含量变化，酶联免疫吸附试验（ELISA）检测血清干扰素γ（IFN-γ）、白介素（IL）-2、IL-17、IL-4、IL-10、转化生长因子β（TGF-β）水平变化。结果肠组织透射电镜超微结构显示Model组造模成功；Model+FMT组与Model+5-ASA组微绒毛较为致密，形态正常，杯状细胞数目较多，线粒体轻微肿胀，粗面内质网病变不明显。Model+FMT组Th17细胞含量高于Control组、低于Model组，Model+5-ASA组Th17细胞含量低于Control组、Model组、Model+FMT组；Model+FMT组、Model+5-ASA组Th1细胞含量均分别低于Control组、Model组；Model+FMT组Th2细胞含量低于Control组、高于Model组，Model+5-ASA组Th2细胞含量低于Control组、高于Model组和Model+FMT组；Model+FMT组、Model+5-ASA组Treg细胞含量均分别低于Control组、高于Model组（P<0.05）。Model+5-ASA组IFN-γ细胞含量低于Model组；Model+FMT组、Model+5-ASA组IL-2细胞含量低于Model组；Model+FMT组、Model+5-ASA组IL-17细胞含量均分别低于Control组和Model组，Model+5-ASA组IL-17细胞含量低于Model+FMT组；Model+FMT组IL-4细胞含量低于Control组、高于Model组，Model+5-ASA组IL-4细胞含量高于Model组；Model+FMT组IL-10细胞含量高于Control组和Model组，Model+5-ASA组IL-10细胞含量高于Model组；Model+FMT组、Model+5-ASA组TGF-β细胞含量均分别低于Control组、高于Model组（P<0.05）。结论FMT可改善小鼠溃疡性结肠炎症状，推测其可能是通过调节Th1/Th2细胞平衡、Th17/Treg细胞比例达到治疗的目的。

关键词: 结肠炎, 溃疡性, 粪便微生物群移植, 氨水杨酸, Th1细胞, Th2细胞, Th17细胞, T淋巴细胞, 调节性

Abstract: Background

Ulcerative colitis is listed as one of the modern refractory diseases by the World Health Organization. At present, Western medicine still has many shortcomings in its treatment, and studies have shown that fecal bacterial transplantation (FMT) has a certain effect on it, but the mechanism is not clear.

Objective

FMT was used to treat mouse ulcerative colitis model to verify the efficacy and possible mechanism of FMT.

Methods

From December 2019 to April 2020, 60 mice were divided into normal control group (Control group) , ulcerative colitis model group (Model group) , ulcerative colitis model + fecal bacteria transplantation treatment group (Model+FMT group) and ulcerative colitis model + 5-aminosalicylic acid (5-ASA) treatment group (Model+5-ASA group) by random number table method, each of 15 mice. Control group did not make any intervention; Model group prepared mouse ulcerative colitis model; after successful modeling, the Model+FMT group was given 0.2 ml of fecal bacteria solution per enema; after successful modeling, the Model+5-ASA group was given 0.019 5 g/ml 5-ASA enema. The ultrastructural changes of intestinal tissue through transmission electron microscope, flow cytometric detection of blood helper T cell (Th) -17, Th-1, Th-2, Treg cell content changes, and enzyme-linked immunosorbent assay (ELISA) were observed to detect serum interferon γ (IFN-γ) , interleukin (IL) -2, IL-17, IL-4, IL-10, transforming growth factor β (TGF-β) level changes.

Results

Intestinal tissue transmission electron microscopy ultrastructure showed that the Model group was successfully modeled; the microvilli in the Model+FMT group and Model+5-ASA group were denser, with normal morphology, more goblet cells, slight swelling of mitochondria, and insignificant rough endoplasmic reticulum lesions. The Th17 cell content of the Model+FMT group was higher than that of the Control group and lower than that of the Model group; the Th17 cell content of the Model+5-ASA group was lower than that of Control group, Model group and Model+FMT group. The Th1 cell content of Model+FMT group and Model+5-ASA group were lower than those of Control group and Model group, respectively; Th2 cell content of Model+FMT group was lower than that of Control group and higher than that of Model group, and Th2 cell content of Model+5-ASA group was lower than that of Control group and higher than that of Model group and Model +FMT group. Treg cell content in Model+FMT group and Model+5-ASA group were lower than that of Control group and higher than that of Model group (P<0.05) . IFN-γ cell content in Model+5-ASA group was lower than that of Model group. IL-2 cell content in Model+FMT group and Model+5-ASA group was lower than that of Model group; the IL-17 cell content of Model+FMT group and Model+5-ASA group were lower than those of Control group and Model group, respectively. The IL-17 cell content of Model+5-ASA group was lower than that of Model+FMT group; the IL-4 cell content of Model+FMT group was lower than that of Control group and higher than that of Model group. The IL-4 cell content in the Model+5-ASA group was higher than that in the Model group; the IL-10 cell content in the Model+FMT group was higher than that in the Control group and the Model group, and the IL-10 cell content in the Model+5-ASA group was higher than that in the Model group; the content of TGF-β cells in the Model+FMT group and Model+5-ASA group were lower than those in the Control group and higher than those in the Model group (P<0.05) .

Conclusion

FMT can improve the symptoms of ulcerative colitis in mice. It is speculated that it may be achieved by adjusting the balance of Th1/Th2 cells and the ratio of Th17/Treg cells to achieve the purpose of treatment.

Key words: Colitis, ulcerative, Fecal microbiota transplantation, Mesalamine, Th1 cells, Th2 cells, Th17 cells, T-lymphocytes, regulatory

中图分类号:

R574.62

翁剑锋,徐佳,刘朋,等. 粪菌移植干预治疗溃疡性结肠炎的免疫学机制研究[J]. 中国全科医学, 2022, 25(03): 298-304. DOI: 10.12114/j.issn.1007-9572.2021.02.069.

WENG Jianfeng, XU Jia, LIU Peng, GE Wei, CHEN Jiaohua, PENG Yingying, HU Jiali, CUI Manman, ZHANG Leichang.

Immunological Mechanism of Faecal Bacterial Transplantation in the Intervention of Ulcerative Colitis [J]. Chinese General Practice, 2022, 25(03): 298-304.

图/表 4

参考文献 15

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组别	只数	Th17	Th1	Th2	Treg
Control组	15	0.88±0.15	8.86±3.00	7.71±1.30	5.94±1.24
Model组	15	5.60±1.00^a	28.20±3.30^a	1.76±1.01^a	0.97±0.51^a
Model+FMT组	15	1.77±0.30^ab	12.50±2.22^ab	3.70±1.61^ab	3.64±1.28^ab
Model+5-ASA组	15	0.47±0.12^abc	11.09±2.07^ab	4.65±1.21^abc	4.20±0.85^ab
F值		189.191	160.214	54.751	61.371
P值		<0.001	<0.001	<0.001	<0.001

组别	只数	Th17	Th1	Th2	Treg
Control组	15	0.88±0.15	8.86±3.00	7.71±1.30	5.94±1.24
Model组	15	5.60±1.00^a	28.20±3.30^a	1.76±1.01^a	0.97±0.51^a
Model+FMT组	15	1.77±0.30^ab	12.50±2.22^ab	3.70±1.61^ab	3.64±1.28^ab
Model+5-ASA组	15	0.47±0.12^abc	11.09±2.07^ab	4.65±1.21^abc	4.20±0.85^ab
F值		189.191	160.214	54.751	61.371
P值		<0.001	<0.001	<0.001	<0.001

组别	只数	IFN-γ	IL-2	IL-17	IL-4	IL-10	TGF-β
Control组	9	147.98±22.57	57.42±10.37	6.01±0.95	23.48±2.62	97.49±5.56	130.79±17.55
Model组	9	183.65±27.12^a	73.87±14.64^a	12.56±1.73^a	10.48±1.73^a	57.93±11.46^a	76.30±14.12^a
Model+FMT组	9	160.33±43.92	49.57±12.24^b	4.36±0.89^ab	20.35±2.39^ab	118.00±10.85^ab	104.88±11.63^ab
Model+5-ASA组	9	142.31±33.65^b	52.17±7.59^b	2.96±0.87^abc	22.00±4.26^b	109.84±22.82^b	98.88±11.87^ab
F值		2.811	8.089	119.367	36.873	31.883	23.051
P值		0.048	<0.001	<0.001	<0.001	<0.001	<0.001

组别	只数	IFN-γ	IL-2	IL-17	IL-4	IL-10	TGF-β
Control组	9	147.98±22.57	57.42±10.37	6.01±0.95	23.48±2.62	97.49±5.56	130.79±17.55
Model组	9	183.65±27.12^a	73.87±14.64^a	12.56±1.73^a	10.48±1.73^a	57.93±11.46^a	76.30±14.12^a
Model+FMT组	9	160.33±43.92	49.57±12.24^b	4.36±0.89^ab	20.35±2.39^ab	118.00±10.85^ab	104.88±11.63^ab
Model+5-ASA组	9	142.31±33.65^b	52.17±7.59^b	2.96±0.87^abc	22.00±4.26^b	109.84±22.82^b	98.88±11.87^ab
F值		2.811	8.089	119.367	36.873	31.883	23.051
P值		0.048	<0.001	<0.001	<0.001	<0.001	<0.001

粪菌移植干预治疗溃疡性结肠炎的免疫学机制研究

Immunological Mechanism of Faecal Bacterial Transplantation in the Intervention of Ulcerative Colitis

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