肝外胆管癌患者肠道菌群变化分析

李涛; 王盼梁; 袁梓博; 冯泉凯; 李捷

doi:10.3969/j.issn.1001-5256.2021.12.029

肝外胆管癌患者肠道菌群变化分析

DOI: 10.3969/j.issn.1001-5256.2021.12.029

李涛^{1, 2, 3},
王盼梁^{2, 3},
袁梓博^{1, 2, 3},
冯泉凯^{1, 2, 3},
李捷^{1, 2, 3, ,}

1.
郑州大学第一附属医院肝胆胰外科, 郑州 450052
2.
河南省消化器官移植重点实验室, 郑州 450052
3.
郑州市器官移植技术与应用工程重点实验室, 郑州 450052

基金项目:

国家自然科学基金 (81900596)

详细信息

通信作者:
李捷，doctorlj100@126.com

中图分类号: R735.8
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出版历程
- 收稿日期: 2021-07-30
- 录用日期: 2021-08-31
- 出版日期: 2021-12-20

Changes in intestinal flora in patients with extrahepatic cholangiocarcinoma

Tao LI^{1, 2, 3},
Panliang WANG^{2, 3},
Zibo YUAN^{1, 2, 3},
Quankai FENG^{1, 2, 3},
Jie LI^{1, 2, 3
, ,}

1.
Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
2.
Henan Key Laboratory of Digestive Organ Transplantation, Zhengzhou 450052, China
3.
Zhengzhou Key Laboratory of Organ Transplantation Technology and Application Engineering, Zhengzhou 450052, China

Research funding:

National Natural Science Foundation of China (81900596)

摘要

摘要: 目的研究肝外胆管癌(ECC)患者肠道菌群变化及影响因素。方法收集2020年1月—2020年12月于郑州大学第一附属医院肝胆胰外科住院治疗的16例ECC患者(ECC组)的粪便样本，采用菌群16S rRNA绝对定量测序，与胆总管结石患者(CBDS组)(20例)及健康对照(Normal组)(10例)进行比较，分析3组肠道菌群差异及与临床指标的相关性。符合正态分布且满足方差齐性的2组间比较采用t检验，3组之间比较采用单因素方差分析；非正态分布3组之间比较采用Kruskal-Wallis H秩和检验，组间比较采用Nemenyi检验。计数资料组间比较采用χ²检验或Fisher确切概率法，相关性检验采用Spearman相关性分析。结果 ECC组TBil和DBil明显高于CBDS组和Normal组(P值均＜0.05)。多样性分析显示Observed species、Chao1指数、Shannon指数在3组之间差异无统计学意义(P值均＞0.05)；而Shannon指数和Simpson指数在3组之间差异有统计学意义，ECC组物种多样性与Normal组相似，但高于CBDS组(P＜0.05)，CBDS组高于Normal组(P＜0.05)。多样性分析显示ECC组菌群结构明显不同于Normal组和CBDS组(P＜0.05)。菌群组成差异性分析表明，Prevotella、Lactobacillus、Megasphaera、Sutterella在ECC组中明显富集。相关分析显示，Prevotella与抗生素、抑酸药和保肝药的使用呈负相关，Lactobacillus、Megasphaera、Sutterella与TBil和DBil呈正相关。结论 ECC组患者肠道菌群发生了明显改变，且菌群改变与ECC患者肝功能和药物的使用密切相关。
- 肝外胆管癌 /
- 胃肠道微生物组 /
- 绝对定量测序
Abstract: Objective To investigate the changes in intestinal flora in patients with extrahepatic cholangiocarcinoma (ECC) and related influencing factors. Methods Fecal samples were collected from 16 patients with ECC who were hospitalized and treated in Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, from January to December 2020, and absolute quantitative bacterial 16S rRNA was used for sequencing. A comparison was made with 20 patients with common bile duct stones (CBDS group) and 10 healthy controls (normal group), and the three groups were compared in terms of the differences in intestinal flora and the association with clinical indices. A one-way analysis of variance was used for comparison of normally distributed data with homogeneity of variance between the three groups, the t-test was used for comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed data between the three groups, and the Nemenyi test was used for comparison between groups. The chi-square test or the Fisher's exact test were used for comparison of categorical data between the three groups, and a Spearman correlation analysis was used to investigate correlation. Results The ECC group had significantly higher levels of total bilirubin (TBil) and direct bilirubin (DBil) than the CBDS group and the normal group. The α diversity analysis showed that there were no significant differences in observed species, Chao1 Index, and Shannon Index between the three groups (all P > 0.05), while there were significant differences in Shannon Index and Simpson Index between the three groups. The ECC group had a similar species diversity to the normal group and a significantly greater species diversity than the CBDS group (P < 0.05), and the CBDS group had a significantly greater species diversity than the normal group (P < 0.05). The β diversity analysis showed that the structure of intestinal flora in the ECC group was significantly different from that in the normal group and the CBDS group (P < 0.05). The analysis of the difference in bacterial composition showed that Prevotella, Lactobacillus, Megasphaera, and Sutterella were significantly enriched in the ECC group. The correlation analysis showed that Prevotella was negatively correlated with the use of antibiotics, acid inhibitors, and liver-protecting drugs, and Lactobacillus, Megasphaera, and Sutterella were positively correlated with TBil and DBil. Conclusion There is a significant change in intestinal flora in patients with ECC, which is closely associated with liver function and the use of drugs.
- Extrahepatic Cholangiocarcinoma /
- Gastrointestinal Microbiome /
- Absolute Quantitative Sequencing

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图 1 稀释性曲线

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图 2 组间PCoA分析

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图 3 3组差异菌属鉴定

注：*，与Normal组比较，P＜0.05；#，与CBDS组比较，P＜0.05。

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图 4 3组菌群进化分支图和LEfSe分析

注: a，不同的颜色表示不同分组, 除黄色节点表示的是在组间绝对定量并无显著差异的物种外, 树枝中其他3种颜色节点表示在对应组别中绝对定量显著高的物种, 节点直径大小与丰度大小呈正比; b，不同颜色的条形表示不同组别中LDA score(log₁₀) 大于2且在该组中丰度显著高的差异物种, 条形图的长短代表的是LDA score值大小。

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表 1 3组一般资料比较

项目	ECC组(n=16)	CBDS组(n=20)	Normal组(n=10)	统计值	P值
男/女(例)	7/9	7/13	5/5	χ²=0.679	0.891
年龄(岁)	61.38±10.69	60.25±16.07	53.20±11.55	F=1.257	0.295
TBil(mol/L)	176.59(63.56~324.11)	29.24(14.74~113.73)¹⁾	14.10(12.23~15.78)¹⁾	χ²=22.471	＜0.001
DBil(mol/L)	150.16(55.11~278.45)	20.79(10.40~101.64)¹⁾	6.96(6.18~8.35)¹⁾	χ²=24.575	＜0.001
ALP(U/L)	268.65(200.25~437.00)	155.00(67.75~319.00)¹⁾	77.50(68.25~89.00)¹⁾	χ²=18.447	＜0.001
GGT(U/L)	379.00(226.50~774.75)	188.5(83.00~411.5)¹⁾	33.00(21.00~39.50)¹⁾	χ²=23.311	＜0.001
ALT(U/L)	80.50(48.25~102.98)	59.00(39.53~123.75)	21.50(18.75~29.00)¹⁾	χ²=17.767	＜0.001
AST(U/L)	67.00(41.50~100.75)	46.00(27.25~85.00)	23.00(18.25~29.25)¹⁾	χ²=15.188	0.001
使用抑酸药天数(d)	13.00±10.67	16.54±8.25		t=0.290	0.885
使用保肝药天数(d)	12.00±5.38	6.60±9.11		t=0.843	0.724
使用抗生素天数(d)	12.33±18.06	14.77±2.34		t=0.621	0.967
PTCD治疗(例)	2	0			0.190
注: 与ECC组比较, 1)P＜0.05。

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表 2 3组间菌群多样性的比较

指数	ECC组(n=16)	CBDS组(n=20)	Normal组(n=10)	F值	P值
Observed species	243±51	211±87	240±68	0.420	0.403
Chao1	244.43±51.59	213.24±88.55	240.98±67.62	0.420	0.421
Shannon	3.50±0.44¹⁾	2.48±0.99	3.37±0.38¹⁾	0.007	0.002
ACE	244.69±51.78	213.53±88.37	241.39±67.65	0.420	0.407
Simpson	0.072±0.041¹⁾	0.259±0.210	0.091±0.176¹⁾	0.002	＜0.001
注: 与CBDS组比较，1)P＜0.05。

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