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.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2022 Aug 1;51(4):438-453. doi: 10.3724/zdxbyxb-2022-0283.
To explore the characteristics of intestinal microecology in hepatocellular carcinoma (HCC) model mice.
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 week after birth. Mice in each group were randomly selected and sacrificed at 10 , 18 and 32 weeks after birth, respectively, the liver tissue samples were obtained for histopathological examination. At the 32 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.
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 <0.05). Beta diversity analysis showed that PCoA based on weighted or unweighted Unifrac distances all yielded >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 ( <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 ( <0.01), while the abundance of Patescibacteria was significantly increased ( <0.05). Moreover, the dominant taxa at the genus level in normal control group mainly included , , , , . The dominant taxa at the genus level in HCC model group mainly included , , , , . There were 30 genera with statistically significant differences in relative abundance at the genus level between the two groups (all <0.05). LEfSe analysis of the intestinal flora of mice in the two groups revealed a total of 14 multi-level differential taxa (all <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 including , , 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, <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 (both <0.05), while that of gram negative ( <0.05) and pathogenic potential ( <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 (all <0.005), including those related to energy metabolism, cell division, nucleotide metabolism, etc., while 12 metabolic pathways were enriched in HCC model group (all <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 , , and could be closely associated with DEN-induced primary HCC in mice.
探索肝癌（HCC）模型小鼠肠道微生物组的特征。
将 2 周龄 C57BL/6 雄性小鼠分为正常对照组和 HCC 模型组。HCC 模型组小鼠于出生后 2 周接受单次腹腔注射二乙基亚硝胺（DEN）；从出生后第 4 周开始，存活的小鼠每隔 2 周腹腔注射 1,4-双[2-（3,5-二氯吡啶氧基）]苯（TCPOBOP），共 8 次。每组随机选择小鼠，分别于出生后 10、18 和 32 周处死，取肝组织标本行组织病理学检查。在第 32 周，两组小鼠全部处死，在处死前无菌条件下采集粪便标本。对 16S rRNA 基因 V3-V4 高变区进行测序，分析物种丰度、菌群多样性和表型，以及菌群相关性和功能预测。
α多样性分析显示，所有 Good's 覆盖率均达到 1.00 的最大值，正常对照组和 HCC 模型组小鼠肠道菌群的 Observed features、Chao1 指数、Shannon 指数和 Simpson 指数差异均有统计学意义（均<0.05）。β多样性分析显示，基于加权或非加权 Unifrac 距离的 PCoA 均产生>0，证实样本内组间差异小于组间差异；两组间分离趋势显著（<0.05）。厚壁菌门、拟杆菌门、放线菌门和 Patescibacteria 是正常对照组和 HCC 模型组的优势门。然而，与正常对照组相比，HCC 模型组中厚壁菌门的丰度显著降低（<0.01），而 Patescibacteria 的丰度显著增加（<0.05）。此外，正常对照组的优势属主要包括、、、、。HCC 模型组的优势属主要包括、、、、。两组间在属水平上有 30 个相对丰度差异有统计学意义的属（均<0.05）。对两组小鼠肠道菌群进行 LEfSe 分析，共发现 14 个多水平差异菌群（均<0.05，LDA 评分>4.0），主要富集于厚壁菌门。正常对照组富集了 10 个差异菌群，包括厚壁菌门、拟杆菌纲、拟杆菌目、穆里巴科等，HCC 模型组富集了 4 个差异菌群，包括、等。正常对照组的优势肠道属之间存在正、负相关（|rho|>0.5，<0.05），而 HCC 模型组的优势肠道属之间的相关性则较为简单，均为正相关。HCC 模型组小鼠肠道菌群中革兰阳性菌和含移动元件的相对丰度显著上调（均<0.05），而革兰阴性菌（<0.05）和致病潜能（<0.05）则显著下调。两组小鼠肠道菌群的代谢途径存在显著差异。例如，正常对照组有 18 个代谢途径富集（均<0.005），包括与能量代谢、细胞分裂、核苷酸代谢等相关的代谢途径，而 HCC 模型组有 12 个代谢途径富集（均<0.005），包括与能量代谢、氨基酸代谢、碳水化合物代谢等相关的代谢途径。结论：DEN 诱导的原发性 HCC 模型小鼠肠道菌群数量减少，小鼠肠道菌群的组成、相关性、表型和功能发生显著改变。厚壁菌门以及、、和等几个属水平的微生物类群可能与 DEN 诱导的原发性 HCC 小鼠密切相关。
