应用16S rDNA扩增子序列分析去卵巢骨丢失小鼠口腔菌群的变化

陈浩彬; 罗世城; 曹祚; 张攻孜; 张书威; 隋熠; 盛晨; 石斌; 罗杨; 张立海

doi:10.3969/j.issn.2095-5227.2023.04.014

应用16S rDNA扩增子序列分析去卵巢骨丢失小鼠口腔菌群的变化

陈浩彬^{1, 2,},
罗世城^{1, 2},
曹祚^{1, 2},
张攻孜^{1, 2},
张书威^{1, 2},
隋熠^{1, 2},
盛晨¹,
石斌^{2, 3},
罗杨^{2, 3},
张立海^{1, 2, 3, ,}

1.
解放军医学院，北京　100853
2.
解放军总医院第一医学中心骨科，北京　100853
3.
解放军总医院第四医学中心骨科医学部，北京　100048

基金项目: 国家重点研发计划(2020YFC2004902)

详细信息

作者简介:
陈浩彬，男，在读硕士，医师。研究方向：骨折与骨质疏松。Email: 1442859440@qq.com

通讯作者:
张立海，男，博士，主任医师，教授，科主任。Email: zhanglihai74@qq.com

中图分类号: R781.2
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出版历程
- 收稿日期: 2022-09-06
- 网络出版日期: 2023-04-13
- 刊出日期: 2023-04-27

Oral microbiota changes of bone loss mice induced by ovariectomy via 16S rDNA amplicon sequencing

CHEN Haobin^{1, 2,},
LUO Shicheng^{1, 2},
CAO Zuo^{1, 2},
ZHANG Gongzi^{1, 2},
ZHANG Shuwei^{1, 2},
SUI Yi^{1, 2},
SHENG Chen¹,
SHI Bin^{2, 3},
LUO Yang^{2, 3},
ZHANG Lihai^{1, 2, 3, ,}

1.
Chinese PLA Medical School, Beijing 100853, China
2.
Department of Orthopedics, the First Medical Center, Chinese PLA General Hospital, Beijing 100853, China
3.
Senior Department of Orthopedics, the Fourth Medical Center, Chinese PLA General Hospital, Beijing 100048, China

More Information

Corresponding author:
ZHANG Lihai. Email: zhanglihai74@qq.com

摘要

摘要:
背景口腔微生物失调加重了多种口腔疾病和系统性疾病的进程。进入绝经后期的妇女罹患口腔黏膜病和加速牙周骨质丢失的风险更高，但口腔菌群变化与女性进入绝经后期之间的联系尚不清楚。
目的应用16S rDNA扩增子测序，初步观察卵巢切除小鼠的口腔菌群结构变化。
方法 16只雌性C57BL/6J小鼠随机分为卵巢切除(ovariectomy，OVX)组和假手术(sham-operated，Sham)组。卵巢切除术4周后对股骨进行显微CT扫描和骨密度与骨参数的分析，取小鼠口腔拭子进行16S rDNA高通量测序。
结果与Sham组比较，OVX组骨密度、骨体积分数、骨小梁厚度和骨小梁数量显著下降，骨表面积/骨体积和骨小梁分离度显著升高(P＜0.05)。两组的口腔菌群共同含有687种ASVs，Sham组特有1086种，OVX特有930种。OVX组的Chao1指数与Sham组差异无统计学意义，但Simpson指数显著低于Sham组(P＜0.05)。门水平上OVX组的拟杆菌门和梭杆菌门丰度较Sham组下降(P＜0.05)。属水平上OVX组的梭杆菌属、乳酸乳球菌属、萨特氏菌属等菌属的丰度相比SHAM组降低(P＜0.05)；放线菌属等菌属的丰度相比Sham组升高(P＜0.05)。LEfSe分析结果显示，Sham组厚壁菌门和芽孢杆菌纲(Bacilli)的相对丰度升高(P＜0.05)。
结论卵巢切除小鼠出现骨丢失现象，并且口腔菌群的多样性和菌群结构发生了改变，初步为研究绝经后妇女口腔菌群改变与口腔疾病加重的关系提供依据。
- 16S rDNA序列分析 /
- 绝经后期 /
- 口腔 /
- 微生物群 /
- 绝经后骨质疏松
Abstract:
Background The dysbiosis of oral microorganisms aggravates the process of several oral diseases and systemic diseases. Postmenopausal women are at increased risk of oral mucosal disease and accelerated periodontal bone loss, but the association between changes in oral microbiota and postmenopause in women is unclear.
Objective To explore the changes of oral microbiota in ovariectomized mice via 16S rDNA amplicon sequencing.
Methods Sixteen female C57BL/6J mice were randomly divided into Ovariectomy group (OVX) and sham-operated group (Sham). The femurs were taken for MicroCT scanning and bone mineral density and bone parameters analysis at 4 weeks after the operation, and the oral swabs samples were collected for 16S rDNA high-throughput sequencing.
Results The bone mineral density, bone volume/total volume, trabecular thickness and trabecular number were decreased, while the bone surface area/bone volume and bone trabecular spacing were increased in OVX group (P<0.05). Two groups of oral samples contained common 687 ASVs, OVX group contained specific 1086 ASVs and OVX group contained specific 930 ASVs. There was no significant difference in Chao1 index between OVX group and Sham group, Simpson index in OVX group was significantly lower than that in Sham group (P<0.05). At the phylum level of oral samples, the relative abundance of Fusobacteriota and Bacteroidota in OVX group was decreased (P<0.05). At the genus level, the relative abundance of Fusobacterium, Lactococcus, Sutterella and other genera in OVX group were decreased, Actinomyces and other genera were increased (P<0.05). LEfSe analysis showed that the relative abundance of Firmicutes and Bacilli increased in the Sham group (P<0.05).
Conclusion Bone loss occurs in ovariectomized mice, and the diversity and structure of oral microbiota are changed, which preliminarily provides reference for the study of the relationship between the change of oral microbiota and the aggravation of oral diseases in postmenopausal women.
- 16S rDNA sequence analysis /
- postmenopause /
- mouth /
- microbiota /
- postmenopausal osteoporosis

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图 1 卵巢切除后小鼠股骨的显微CT结果

A：骨密度(BMD)；B：骨体积分数(BV/TV)；C：骨表面积/骨体积(BS/BV)；D：骨小梁厚度(Tb.Th)；E：骨小梁数量(Tb.N)；F：骨小梁分离度(Tb.Sp)；G：股骨显微CT扫描图像

Figure 1. MicroCT results of the femur of mice after ovariectomy

A: Bone mineral density (BMD); B: Bone volume/total volume (BV/TV); C: Bone surface area/bone volume (BS/BV); D: Trabecular thickness (Tb.Th); E: Trabecular number (Tb.N); F: Trabecular Spacing (Tb.Sp); G: MicroCT scan of femur

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图 2 口腔菌群的ASVs分析韦恩图

Figure 2. Venn diagram of ASVs analysis of oral microbiota

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图 3 口腔菌群各组门水平和属水平物种相对丰度柱形图

A：门水平相对丰度；B：属水平相对丰度

Figure 3. Relative abundance bar chart of species at phylum level and genus level for each group of oral microbiota

A: Relative abundance at phylum level; B: Relative abundance at genus level

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图 4 各组样本菌群丰度和菌群多样性差异

A: Chao1指数差异箱型图；B: Simpson指数差异箱型图

Figure 4. The differences of microbiota abundance and microbiota diversity between each group

A: Box diagram of Chao1 index difference; B: Box diagram of Simpson index difference

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图 5 口腔各组样本的PCA分析图

Figure 5. Principal components analysis (PCA) of oral species between each group

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图 6 口腔各组物种相对丰度差异分析

A：门水平物种相对丰度差异；B：纲水平物种相对丰度差异；C：目水平物种相对丰度差异；D：科水平物种相对丰度差异；E：属水平物种相对丰度差异

Figure 6. Differences in relative abundance of oral species among different groups

A: Differences in relative abundance at phylum level; B: Differences in relative abundance of species at class level; C: Differences in relative abundance of species at order level; D: Differences in relative abundance of species at family level; E: Differences in relative abundance of species at genus level species

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图 7 口腔各组样本的LEfSe分析

Figure 7. LEfSe analysis of oral species between each group

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