二乙基亚硝胺诱导肝细胞癌模型鼠肠道微生态研究

doi:10.3724/zdxbyxb-2022-0283

浙江大学学报（医学版）

2022, Vol. 51

Issue (4): 438-453 DOI: 10.3724/zdxbyxb-2022-0283

原著

二乙基亚硝胺诱导肝细胞癌模型鼠肠道微生态研究

周文斌¹,郑越^2,^3,⁴,尚佳^2,^3,⁴,王海洋^2,^3,⁴,王依莎^2,^3,⁴,陆欢^2,^3,⁴,王小西⁵,隋梅花^2,^3,^4,*(

)

1. 青岛大学青岛医学院，山东青岛 266071
2. 浙江大学医学院基础医学院，浙江杭州 310058
3. 浙江大学医学院附属妇产科医院，浙江杭州 310006
4. 浙江大学癌症研究院，浙江杭州 310058
5. 浙江大学医学院附属第一医院病理科，浙江杭州 310003

Intestinal microecology in mice bearing diethylnitrosamine-induced primary hepatocellular carcinoma

ZHOU Wenbin¹,ZHENG Yue^2,^3,⁴,SHANG Jia^2,^3,⁴,WANG Haiyang^2,^3,⁴,WANG Yisha^2,^3,⁴,LU Huan^2,^3,⁴,WANG Xiaoxi⁵,SUI Meihua^2,^3,^4,*(

)

1. Qingdao Medical College, Qingdao University, Qingdao 266071, Shandong Province, China;
2. School of Basic Medical Sciences, Zhejiang University School of Medicine, Hangzhou 310058, China;
3. Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China;
4. Zhejiang University Cancer Center, Hangzhou 310058, China;
5. Department of Pathology, the First Affiliated Hospital, Zhejiang University School Medicine, Hangzhou 310003, China

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摘要：

目的：探索肝细胞癌模型鼠肠道微生态特征。方法：将2周龄C57BL/6雄鼠分为正常对照组和肝癌模型组。肝癌模型组在出生后2周给予单次腹腔注射二乙基亚硝胺（DEN），其中存活小鼠自4周龄起腹腔注射1，4-双[2-（3，5-二氯吡啶氧）]苯，每2周一次，共8次。分别于小鼠出生后第10、18和32周随机处死部分实验动物，取肝脏进行组织病理学检查。其中，在小鼠出生后第32周处死动物前无菌条件下收集两组粪便标本，并进行V3~V4高可变区16S rRNA基因组测序，进而对菌群多样性、物种丰度差异、菌群相关性、表型预测以及功能预测进行分析。结果：α多样性分析结果显示Good’s coverage均已达到最大值1.00，且正常对照组与肝癌模型组肠道菌群Observed features、Chao1指数、Shannon指数和Simpson指数差异均有统计学意义（均 P<0.05）。β多样性分析结果显示基于加权或未加权Unifrac距离的主坐标PCoA均R>0，标本的组内差异小于组间差异；两组标本的分离趋势明显（P<0.05）。拟杆菌门、厚壁菌门、放线菌门及髌骨菌门等菌群为正常对照组与肝癌模型组门水平的优势菌群，但肝癌模型组较正常对照组拟杆菌门丰度显著减少（P<0.01），而髌骨菌门丰度显著增加（P<0.05）。正常对照组属水平的优势菌群主要为鼠杆菌科未分类属、副鼠杆菌属、鼠杆菌属、毛螺旋菌科NK4A136属、欧陆森氏菌属等，而肝癌模型组为艾克曼菌属、杜氏杆菌属、鼠杆菌科未分类属、毛螺旋菌科NK4A136属、红椿杆菌科UCG-002属等。两组属水平菌群相对丰度差异有统计学意义的有30个菌属（均P<0.05）。两组小鼠肠道菌群LEfSe分析共发现14个多级别差异物种（均P<0.05，LDA评分超过4.0），主要富集在拟杆菌门。其中正常对照组为拟杆菌门、拟杆菌纲、拟杆菌目、鼠杆菌科等10个差异菌群富集，肝癌模型组为杜氏杆菌属、消化链球菌属等4个差异菌群富集。正常对照组肠道优势菌属之间既有正相关关系又有负相关关系（|rho|>0.5，P<0.05），肝癌模型组肠道优势菌属的相关性网络复杂程度比正常对照组降低，所有优势菌群的相互作用均呈较强的正相关。与正常对照组比较，肝癌模型组肠道菌群革兰阳性菌及移动元件菌基因相对丰度均显著上调（均P<0.05），而革兰阴性菌（P<0.05）及潜在致病性菌基因（P<0.05）相对丰度均显著下调。两组肠道菌群的基因代谢通路差异显著，其中正常对照组富集在能量代谢、细胞分裂及核苷酸代谢等18个代谢通路，而肝癌模型组富集在能量代谢、氨基酸代谢及碳水化合物代谢等12个代谢通路（均P<0.005）。结论：DEN诱导原发性肝癌模型小鼠肠道菌群数减少，菌群构成、菌群相关性、表型和功能均发生改变，其中门水平的拟杆菌门以及属水平的鼠杆菌科未分类属、鼠杆菌属、消化链球菌属、杜氏杆菌属等菌群可能与DEN诱导肝细胞癌的发生有关。

关键词： 肝细胞癌; 肠道菌群; 物种多样性; 菌群构成; 16S?核糖体RNA测序; 二乙基亚硝胺; C57BL/6小鼠

Abstract:

Objective: To explore the characteristics of intestinal microecology in hepatocellular carcinoma (HCC) model mice. Methods: C57BL/6 male mice aged 2 weeks were divided into normal control group and HCC model group. Mice in HCC model group were exposed to a single intraperitoneal injection of diethylnitrosamine (DEN) 2 weeks after birth; the surviving mice were intraperitoneally injected with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), once every 2 weeks for 8 times starting from the 4 ^th week after birth. Mice in each group were randomly selected and sacrificed at 10 ^th, 18 ^th and 32 ^nd weeks after birth, respectively, the liver tissue samples were obtained for histopathological examination. At the 32 ^nd week, all mice in both groups were sacrificed and the feces samples were collected under sterile conditions right before the sacrifice. The feces samples were sequenced for the V3-V4 hypervariable regions of the 16S rRNA gene, and the species abundance, flora diversity and phenotype, as well as flora correlation and functional prediction were analyzed. Results: Alpha diversity analysis showed that all Good’s coverage reached the maximum value of 1.00, and the differences in the Observed features, Chao1 index, Shannon index and Simpson index of the intestinal flora of mice between normal control group and HCC model group were all statistically significant (all P<0.05). Beta diversity analysis showed that PCoA based on weighted or unweighted Unifrac distances all yieldedR>0, confirming that the intra-group differences of the samples were less than the inter-group differences; the trend of separation between the two groups was significant (P<0.05). Bacteroidetes, Firmicutes, Actinobacteria and Patescibacteria were the dominant taxa at the phylum level in both normal control group and HCC model group. However, compared with normal control group, the abundance of Bacteroidetes in HCC model group was significantly decreased (P<0.01), while the abundance of Patescibacteria was significantly increased (P<0.05). Moreover, the dominant taxa at the genus level in normal control group mainly includedMuribaculaceae_unclassified, Paramuribaculum, Muribaculum, Lachnospiraceae_NK4A 136 group, Olsenella. The dominant taxa at the genus level in HCC model group mainly included Akkermansia, Dubosiella, Muribaculaceae_unclassified, Lachnospiraceae_NK4A 136 group, Coriobacteriaceae_UCG-002. There were 30 genera with statistically significant differences in relative abundance at the genus level between the two groups (all P<0.05). LEfSe analysis of the intestinal flora of mice in the two groups revealed a total of 14 multi-level differential taxa (allP<0.05, LDA score>4.0), which were mainly enriched in Bacteroidetes. The enrichment of 10 differential taxa including Bacteroidetes, Bacteroidia, Bacteroidales, Muribaculaceae, etc. were found in normal control group, and the enrichment of 4 differential taxa includingDubosiella, Peptostreptococus, etc. were found in HCC model group. There were both positive and negative correlations between the dominant intestinal genera in normal control group (|rho|>0.5,P<0.05), while the correlations of the dominant intestinal genera in HCC model group, being less complex than that in normal control group, were all positive. The relative abundance of gram positive and mobile element containing in the intestinal flora of mice in HCC model group was significantly up-regulated compared with normal control group (bothP<0.05), while that of gram negative (P<0.05) and pathogenic potential (P<0.05) was significantly down-regulated. The metabolic pathways of the intestinal flora in the two groups were significantly different. For instance, 18 metabolic pathways were enriched in normal control group (allP<0.005), including those related to energy metabolism, cell division, nucleotide metabolism, etc., while 12 metabolic pathways were enriched in HCC model group (allP<0.005), including those related to energy metabolism, amino acid metabolism, carbohydrate metabolism, etc.Conclusions: The amount of intestinal flora in DEN-induced primary HCC model mice decreased, and the composition, correlation, phenotype and function of the intestinal flora in mice were significantly altered. Bacteroidetes at the phylum level, as well as several microbial taxa at the genus level such as Muribaculaceae_unclassified, Muribaculum, Peptostreptococus and Dubosiella could be closely associated with DEN-induced primary HCC in mice.

Key words: Hepatocellular carcinoma Intestinal flora Species diversity Microbial community structure 16S rRNA sequencing Diethylnitrosamine C57BL/6 mouse

收稿日期: 2022-05-31 出版日期: 2022-11-16

CLC:

R735.7

基金资助: 国家自然科学基金（21722405，22075243）; 浙江大学“百人计划”研究员启动基金

通讯作者: 隋梅花 E-mail: suim@zju.edu.cn

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引用本文:

周文斌,郑越,尚佳,王海洋,王依莎,陆欢,王小西,隋梅花. 二乙基亚硝胺诱导肝细胞癌模型鼠肠道微生态研究[J]. 浙江大学学报（医学版）, 2022, 51(4): 438-453.

ZHOU Wenbin,ZHENG Yue,SHANG Jia,WANG Haiyang,WANG Yisha,LU Huan,WANG Xiaoxi,SUI Meihua. Intestinal microecology in mice bearing diethylnitrosamine-induced primary hepatocellular carcinoma. J Zhejiang Univ (Med Sci), 2022, 51(4): 438-453.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0283 或 https://www.zjujournals.com/med/CN/Y2022/V51/I4/438

图1 DEN诱导小鼠肝细胞癌的建模流程示意图 TCPOBOP：1,4-双[2-(3,5-二氯吡啶氧)]苯.

图2 正常对照组与肝癌模型组10、18和32周龄时肝脏大体和组织病理学变化大体形态学观察可见，正常对照组在三个时间点下肝脏均形态正常、表面光滑、边缘锐利，呈正常的红褐色；肝癌模型组10、18周龄时肝脏大体形态与正常对照组相似，但32周龄时肉眼可见肝脏表面出现多个散在、圆形或椭圆形、大小不等的癌结节. 病理学检查可见正常对照组在三个时间点肝脏细胞形态正常，排列整齐有序，无脂肪变性或异型增生；肝癌模型组10周龄时肝脏可见炎症细胞浸润、细胞肿胀、脂肪变性等，18周龄肝脏可见细胞内玻璃样变、脂肪变性以及细胞间的胶原纤维沉积等表现，32周龄时肝脏可见癌变细胞体积增加，胞浆丰富呈嗜酸性，细胞核异型性明显，核仁增大并可见病理性核分裂像以及典型的肝纤维化和肝硬化病变.

表1 正常对照组与肝癌模型组10、18和32周龄时体重、肝重及肝体比比较

图3 正常对照组与肝癌模型组肠道微生物β多样性分析结果 A：非加权的UniFrac PCoA图；B：加权的UniFrac PCoA图. 图中每个点代表单个标本. 正常对照组 =5，肝癌模型组 =9.

图4 正常对照组与肝癌模型组肠道菌群门水平构成柱子的长短表示菌群的相对丰度.

表2 正常对照组与肝癌模型组门水平肠道细菌优势菌群相对丰度比较

图5 正常对照组与肝癌模型组肠道菌群属水平构成柱子的长短表示菌群的相对丰度.

组　别	n	异杆菌属	消化链球菌属	红椿杆菌科 UCG-002属	杜氏杆菌属	低嗜盐细菌属	候选单胞生糖菌属
正常对照组	5	0.000(0.000, 0.006)	0.000(0.000, 0.000)	0.714(0.391, 2.397)	1.989(1.607, 11.636)	0.000(0.000, 0.000)	0.281(0.131, 0.308)
肝癌模型组	9	0.135(0.034, 0.217)	0.011(0.005, 0.018)	2.169(1.748, 4.268)	13.592(8.877, 23.362)	0.010(0.000, 0.032)	0.406(0.321, 0.748)
Z值	—	–2.562	–2.492	–2.200	–2.067	–2.215	–2.200
P值	—	<0.01	<0.05	<0.05	<0.05	<0.05	<0.05
组　别	n	瘤胃梭菌属	颤杆菌属	瘤胃菌科 UCG-010属	瘤胃球菌属	臭气杆菌属	瘤胃菌科未分类属
正常对照组	5	0.177(0.160, 0.289)	0.270(0.169, 0.444)	0.303(0.140, 0.405)	0.032(0.008, 0.043)	1.345(1.120, 2.414)	0.305(0.165, 0.437)
肝癌模型组	9	0.033(0.012, 0.063)	0.034(0.004, 0.107)	0.052(0.012, 0.058)	0.000(0.000, 0.000)	0.493(0.394, 0.936)	0.072(0.051, 0.238)
Z值	—	–2.333	–2.202	–3.000	–2.719	–2.200	–2.067
P值	—	<0.05	<0.05	<0.01	<0.05	<0.05	<0.05
组　别	n	瘤胃菌科 UCG-005属	普雷沃菌科 NK3B31属	埃格特菌属	邓氏杆菌属	丹毒丝菌科未分类属	瘤胃菌科 UCG-007属
正常对照组	5	0.017(0.013, 0.020)	0.148(0.050, 0.274)	0.148(0.076, 0.285)	0.163(0.058, 0.191)	0.150(0.061, 0.230)	0.028(0.011, 0.033)
肝癌模型组	9	0.000(0.000, 0.007)	0.000(0.000, 0.000)	0.053(0.032, 0.086)	0.000(0.000, 0.000)	0.025(0.000, 0.046)	0.000(0.000, 0.000)
Z值	—	–2.386	–3.006	–2.200	–3.496	–2.494	–3.496
P值	—	<0.05	<0.05	<0.05	<0.01	<0.05	<0.01
组　别	n	DTU014_ unclassified属	拟杆菌目未分类属	贪噬菌属	另枝菌属	Candidatus_ Stoquefichus属	气单胞菌属
正常对照组	5	0.042(0.014, 0.058)	0.247(0.079, 0.303)	0.007(0.003, 0.012)	2.804(1.861, 5.118)	0.048(0.017, 0.094)	0.028(0.021, 0.043)
肝癌模型组	9	0.000(0.000, 0.000)	0.011(0.005, 0.057)	0.000(0.000, 0.000)	0.574(0.162, 1.183)	0.000(0.000, 0.000)	0.000(0.000, 0.019)
Z值	—	–2.719	–2.603	–3.006	–2.867	–2.719	–2.567
P值	—	<0.05	<0.01	<0.05	<0.01	<0.05	<0.05
组　别	n	Millionella属	乳头杆菌属	鼠杆菌属	普雷沃菌科 UCG-001属	鼠杆菌科未分类属	未分类属
正常对照组	5	0.042(0.032, 0.091)	0.020(0.010, 0.069)	5.836(4.598, 7.920)	0.368(0.178, 0.502)	29.474(23.071, 38.218)	0.650(0.502, 0.779)
肝癌模型组	9	0.000(0.000, 0.000)	0.000(0.000, 0.012)	1.806(1.262, 4.266)	0.024(0.000, 0.123)	9.864(8.893, 22.861)	0.361(0.065, 0.470)
Z值	—	–3.006	–2.349	–2.067	–2.612	–2.333	–2.333
P值	—	<0.05	<0.05	<0.05	<0.01	<0.05	<0.05

表3 正常对照组与肝癌模型组相对丰度差异有统计学意义属水平肠道菌群

图6 正常对照组与肝癌模型组肠道菌群LEfSe分析结果 A：肠道菌群进化分支图，显示与正常对照组或肝癌模型组相关微生物群的系统发育分布，其中由内至外辐射的圆圈代表了由界(单个圆圈)至属(或种)的分类级别，小圆圈直径大小与相对丰度大小呈正比；黄色点为正常对照组与肝癌模型组之间无显著差异的微生物类群，绿色点为在正常对照组中起重要作用的微生物类群，红色点为在肝癌模型组中起重要作用的微生物类群. B：线性判别分析分数直方图. 正常对照组 =5，肝癌模型组 =9.

表4 差异肠道菌群诊断肝癌的ROC曲线分析结果

图7 正常对照组和肝癌模型组丰度前三十位细菌属的相关性网络绿色节点为正常对照组，红色节点为肝癌模型组；节点大小代表中心度，中心度越大，相关对象个数越多；节点之间的连接表明两个属之间存在相关性，相关性系数 |rho|>0.5， < 0.05；线条粗细代表相关性强弱，线条越粗表明相关性越强，而线条越细表明相关性越弱；实线表明正相关，虚线表明负相关.

图8 正常对照组和肝癌模型组各类表型肠道菌群的表达丰度比较组间比较, <0.05.

图9 菌群基因功能预测与代谢通路差异分析根据同源基因簇数据库功能注释结果筛选出差异有统计学意义的功能, 横柱状图代表富集在该代谢通路的丰度分别在正常对照组与肝癌模型组标本中所有代谢通路的百分比.

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