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摘要：
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摘要：目的 基于16S rRNA基因测序技术分析非酒精性脂肪性肝病（non-alcoholic fatty
liver disease，NAFLD）大鼠的肠道菌群。方法 以SPF级SD大鼠为实验对象，随机分为
普通饮食组（对照组）和高脂饮食组（模型组）。高脂饮食组通过进食高脂饲料8周
诱发NAFLD模型。8周后检测大鼠血清丙氨酸氨基转移酶（alanine aminotransferase，
ALT）、天门冬氨酸氨基转移酶（aspartate aminotransferase，AST）、高密度脂蛋白
胆固醇（high-density lipoprotein cholesterol，HDL-C）、低密度脂蛋白胆固醇（low
density lipoprotein cholesterol，LDL-C）、甘油三酯（triglyceride，TG）和总胆固醇
（total cholesterol，TC）水平，取肝组织进行HE及油红O染色，采集大鼠新鲜粪便通
过高通量测序法对肠道粪便细菌16S rRNA的V3-V4区进行基因测序。利用UPARSE软
件进行类单元（operational taxonomic unit，OTU）聚类，采用RDP Classifier算法对OTU
代表序列进行比对分析，并在门、纲、目、科、属、种等水平上进行注释其群落的
物种信息。基于OTU聚类结果，利用mothur软件和Qimme（V.1.8）进行Alpha多样性
分析和Beta多样性分析，并通过LEfSe软件进行组间菌落差异分析。结果 与正常组相
比，模型组大鼠AST [（23.63 ± 4.82）U/L vs （10.61 ± 1.17）U/L]、ALT [（9.98 ± 2.27）U/L
vs （3.40 ± 0.81）U/L]、LDL-C [（0.69 ± 0.11）mmol/L vs （0.34 ± 0.10）mmol/L]、
TG [（0.90 ± 0.24）mmol/L vs （0.33 ± 0.13）mmol/L] 及TC [（5.69 ± 0.72）mmol/L vs
（2.10 ± 0.42）mmol/L] 水平均显著升高，HDL-C [（0.62 ± 0.14）mmol/L vs （1.07 ±
0.17）mmol/L] 水平显著降低，差异均有统计学意义（P均＜ 0.05）。两组共获得
2607个OTU，经过抽平处理后获得2547个OTU，其中对照组2367个，模型组2168个，
两组共享OTU为1988个。随样本量增加物种累积曲线趋于平缓，表明样本量充分。
在Alpha多样性指数中，模型组的香农指数（7.0673 ± 0.4812 vs 6.1695 ± 0.7165）、
物种个数 [（968.6250 ± 233.0221）个 vs （1155.9500 ± 129.0011）个] 和PD_Whole_
Tree指数（69.3449 ± 14.2872 vs 82.0219 ± 10.2012）显著低于对照组，差异有统计学
意义（P均＜ 0.05），Chao1指数也低于对照组（1418.4503 ± 277.8639 vs 1599.1725 ±
100.1048），但差异无统计学意义（P ＞ 0.05）。采用主成分分析、主坐标分析、
层次聚类分析、偏最小二乘法判别分析和非度量多维尺度法等Beta多样性分析方法
均能将模型组与对照组大鼠完全区分开来，进一步说明二者间的菌群有明显区别。
采用RDP Classifier算法对OTU代表序列进行比对分析，结果表明，从门水平分析，
NAFLD大鼠厚壁菌门和放线菌门丰度增加，其中放线菌门丰度显著增加，拟杆菌门
和变形菌门的丰度降低。从纲水平分析，NAFLD大鼠放线菌纲和丹毒丝菌纲相对丰
度显著升高，而杆菌纲相对丰度显著下降。从目水平分析，NAFLD大鼠红椿菌目和
丹毒丝菌目相对丰度显著升高，而乳杆菌目和酸微菌目相对丰度显著降低。从科水平
分析，NAFLD大鼠毛螺菌科（Lachnospiraceae）、瘤胃菌科（Ruminococcaceae）、消
化链球菌科（Peptostreptococcus）、丹毒丝菌科（Erysipelotrichaceae）和双歧杆菌科
（Bifidobacteriaceae）等增加，其中双歧杆菌科、肽链球菌科显著增加，乳酸杆菌科
（Lactobacillaceae）、普雷沃氏菌科（Prevotellaceae）和拟杆菌目S24-7组比例下降，
其中乳酸杆菌科显著性减少。采用LEfSe对组间差异显著的物种分析，结果表明两组间
差异起重要作用的肠道微生物类群共126个，LDA值大于3.6的微生物类群共24个。结
论 与普通饮食组相比，高脂饮食组干预8周后大鼠肠道菌群结构和多样性均发生显著变
化。
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Abstract: Objective To analyze the intestinal flora of rats with non-alcoholic fatty liver
disease (NAFLD) by 16S rRNA gene sequencing technology. Methods SPF grade SD rats
were used as experimental subjects and randomly divided into normal diet group (control
group) and high-fat diet group (model group). The high-fat diet group induced NAFLD model
by consuming high-fat feed for 8 weeks. After 8 weeks, serum alanine aminotransferase (ALT),
aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density
lipoprotein cholesterol (LDL-C), triglycerides (TG) and aspartate aminotransferase (AST) of
rats were detected. Liver tissue was taken for HE and oil red O staining, and fresh feces of
rats were collected for gene sequencing of the V3-V4 region of 16S rRNA in intestinal feces
using high-throughput sequencing. UPARSE software was used for operational taxonomic unit
(OTU) clustering, RDP Classifier algorithm was used for alignment analysis of OTU representative
sequences, and annotating species information of their communities at the phylum, class, order,
family, genus, species level. Based on the OTU clustering results, Alpha diversity analysis and
Beta diversity analysis were conducted using Mathur software and Qimme (V.1.8), and inter group
colony differences were analyzed by LEfSe software. Results Compared with rats in normal group,
the levels of AST [(23.63 ± 4.82) U/L vs (10.61 ± 1.17) U/L], ALT [(9.98 ± 2.27) U/L vs (3.40 ±
0.81) U/L], LDL-C [(0.69 ± 0.11) mmol/L vs (0.34 ± 0.10) mmol/L], TG [(0.90 ± 0.24) mmol/L vs (0.33 ±
0.13) mmol/L] and TC [(5.69 ± 0.72) mmol/L vs (2.10 ± 0.42) mmol/L] of rats in model group
increased significantly, the levels of HDL-C [(0.62 ± 0.14) mmol/L vs (1.07 ± 0.17) mmol/L]
reduced significantly, the differences were statistically significant (all P ＜ 0.05). A total
of 2607 OTUs were obtained in two groups, and after leveling treatment, 2547 OTUs were
obtained, including 2367 in control group and 2168 in model group. The two groups shared
1988 OTUs. As the sample size increased, the species accumulation curve tended to flatten
out, indicating that the sample size was sufficient. In the Alpha diversity index, the Shannon
index (7.0673 ± 0.4812 vs 6.1695 ± 0.7165), the number of species (968.6250 ± 233.0221 vs
1155.9500 ± 129.0011), and PD_Whole_Tree index (69.3449 ± 14.2872 vs 82.0219 ± 10.2012)
of rats in model group were significantly lower than those of control group, the differences
were statistically significant (all P ＜ 0.05). The Chao1 index of rats in control group was
lower than that of control group (1418.4503 ± 277.8639 vs 1599.1725 ± 100.1048), but the
difference was not statistically significant (P ＞ 0.05). Beta diversity analysis methods such as
principal component analysis, principal coordinate analysis, hierarchical clustering analysis,
partial least squares discriminant analysis and non metric multidimensional scaling could
completely distinguish the model group from the control group, further indicating a significant
difference in the bacterial community between the two groups. The RDP Classifier algorithm
was used to compare and analyze OTU representative sequences. The results showed that at
the phylum level, the abundance of Firmicutes and Actinobacteria in NAFLD rats increased,
among which Actinobacteria abundance increased significantly, while Bacteroidetes and
Proteobacteria abundance decreased significantly. At the class level, the relative abundance of
Actinobacteria and Erythromycetes in NAFLD rats increased significantly, while the relative
abundance of Bacteroides decreased significantly. From the perspective of order level, the relative
abundance of Coriobacteriales and Erysipelothrix in NAFLD rats increased significantly, while
the relative abundance of Lactobacillales and Acidimicrobiales decreased significantly. From
a family level, Lachnospiraceae, Ruminococcaceae, Peptostreptococcus, Erysipelotrichaceae
and Bifidobacteriaceae increased in NAFLD rats, among which Bifidobacteriaceae and
Peptostreptococcus increased significantly, while Lactobacilliaceae, the proportion of
Prevotellaceae and S24-7 groups of Bacteroideales decreased, among which Lactobacillus
decreased significantly. LEfSe was used to analyze species with significant differences between
the two groups, the results showed that there were 126 gut microbiota groups with significant
differences between the two groups, and 24 microbiota groups with LDA values greater than 3.6.
Conclusions Compared with rats in general diet group, the structure and diversity of intestinal
flora changed significantly in high-fat diet group.
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