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通讯作者:

张国新,E-mail:guoxinz@njmu.edu.cn

中图分类号:R735.2

文献标识码:A

文章编号:1007-4368(2022)12-1651-07

DOI:10.7655/NYDXBNS20221202

参考文献 1
NARDONE G,COMPARE D.The human gastric microbi⁃ ota:Is it time to rethink the pathogenesis of stomach dis⁃ eases[J].United European Gastroenterol J,2015,3(3):255-260
参考文献 2
HUNT R H,YAGHOOBI M.The esophageal and gastric microbiome in health and disease[J].Gastroenterol Clin North Am,2017,46(1):121-141
参考文献 3
ABREU M T,PEEK R M.Gastrointestinal malignancy and the microbiome[J].Gastroenterology,2014,146(6):1534-1546.e3
参考文献 4
周铖,孙鹏飞,解昕轶,等.质子泵抑制剂对消化道菌群组成变化的相关性研究进展[J].南京医科大学学报(自然科学版),2020,40(9):1386-1390
参考文献 5
DICKSVED J,LINDBERG M,ROSENQUIST M,et al.Mo⁃ lecular characterization of the stomach microbiota in pa⁃ tients with gastric cancer and in controls[J].J Med Micro⁃ biol,2009,58(4):509-516
参考文献 6
DI CIAULA A,WANG D Q,MOLINA⁃MOLINA E,et al.Bile acids and cancer:direct and environmental ⁃ depen⁃ dent effects[J].Ann Hepatol,2017,16(Suppl.1:s3 ⁃ 105.):s87-s105
参考文献 7
LEE W,UM J,HWANG B,et al.Assessing the progres⁃ sion of gastric cancer via profiling of histamine,histidine,and bile acids in gastric juice using LC⁃MS/MS[J].J Ste⁃ roid Biochem Mol Biol,2020,197:105539
参考文献 8
QUINN L,SHEH A,ELLIS J L,et al.Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS⁃GAS mice triggers dysbiosis and vitamin K de⁃ ficiency resulting in gastric hemorrhage[J].Gut Mi⁃ crobes,2020,11(4):820-841
参考文献 9
PINZON⁃GUZMAN C,MEYER A R,WISE R,et al.Eval⁃ uation of lineage changes in the gastric mucosa following infection with helicobacter pylori and specified intestinal flora in INS⁃GAS mice[J].J Histochem Cytochem,2019,67(1):53-63
参考文献 10
QUANTE M,BHAGAT G,ABRAMS J A,et al.Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett ⁃like metaplasia[J].Cancer Cell,2012,21(1):36-51
参考文献 11
YUAN T,NI Z,HAN C,et al.SOX2 interferes with the function of CDX2 in bile acid ⁃induced gastric intestinal metaplasia[J].Cancer Cell Int,2019,19:24
参考文献 12
RIVIÈRE A,SELAK M,LANTIN D,et al.Bifidobacteria and butyrate ⁃ producing colon bacteria:importance and strategies for their stimulation in the human gut[J].Front Microbiol,2016,7:979
参考文献 13
TAWTHEP S,FUKIYA S,LEE J Y,et al.Isolation of six novel 7⁃oxo ⁃ or urso ⁃type secondary bile acid ⁃producing bacteria from rat cecal contents[J].J Biosci Bioeng,2017,124(5):514-522
参考文献 14
PARK C H,LEE A R,LEE Y R,et al.Evaluation of gas⁃ tric microbiome and metagenomic function in patients with intestinal metaplasia using 16S rRNA gene sequenc⁃ ing[J].Helicobacter,2019,24(1):e12547
参考文献 15
LOKE M F,LUI S Y,NG B L,et al.Antiadhesive proper⁃ ty of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin[J].FEMS Immunol Med Microbiol,2007,50(2):231-238
参考文献 16
ZAMAN C,OSAKI T,HANAWA T,et al.Analysis of the microbial ecology between Helicobacter pylori and the gastric microbiota of mongolian gerbil[J]s.J Med Micro⁃ biol,2014,63(Pt 1):129-137
参考文献 17
AVILES⁃JIMENEZ F,VAZQUEZ⁃JIMENEZ F,MEDRA⁃ NO ⁃GUZMAN R,et al.Stomach microbiota composition varies between patients with non⁃atrophic gastritis and pa⁃ tients with intestinal type of gastric cancer[J].Sci Rep,2014,4:4202
参考文献 18
FERREIRA R M,PEREIRA ⁃ MARQUES J,PINTO ⁃ RI⁃ BEIRO I,et al.Gastric microbial community profiling re⁃ veals a dysbiotic cancer ⁃ associated microbiota[J].Gut,2018,67(2):226-236
参考文献 19
KANG X,ZHAN L,LU X,et al.Characteristics of gastric microbiota in GK rats with spontaneous diabetes:a compar⁃ ative study[J].Diabetes Metab Syndr Obes,2020,13:1435-1447
目录contents

    摘要

    目的:探究次级胆汁酸脱氧胆酸(deoxycholic acid,DCA)对肠型胃癌模型INS-GAS小鼠胃内菌群结构的影响。方法:将10只8周龄雄性INS-GAS小鼠随机分为2组,每组5只,一组予以0.2% DCA饮水,另一组饮用生理盐水(normal saline, NS)溶剂。另有5只同窝雄性FVB/N野生型小鼠予以0.2% DCA饮水作为基因型对照组。3个月饮水干预结束后收集各组小鼠胃内容物行16S rDNA扩增子测序,进行胃内菌群相对丰度、α多样性指数、β多样性及差异物种分析。分离胃体和胃窦黏膜组织,qRT-PCR检测肠化标志物cdx 2、klf 5、vil 1和muc 2 mRNA表达。结果:与对照组小鼠相比,DCA改变了INS-GAS小鼠胃内优势菌种,属水平上相对丰度最高的菌种由乳杆菌科菌属(61.93%)变为未定义蓝藻细菌属(58.70%)。两组小鼠胃内菌群丰度及多样性均无显著差异(ACE指数P =0.213,Chao1指数P =0.280;Shannon指数P =0.391,Simpson指数P =0.205)。主成分分析发现两组小鼠胃内菌群结构差异度大,组间差异大于组内差异(R =0.368,P =0.032)。DCA饮水促进INS-GAS小鼠胃内蓝藻细菌在门、纲、目、科和属各个水平上显著增高,毛螺菌科Anaerostipes菌属以及两种蓝藻细菌属菌种Lolium perennePhaseolus vulgaris丰度也显著增高。此外,DCA饮水同样导致INS-GAS小鼠胃内厚壁菌门、杆菌纲和乳杆菌(目、科和属水平)丰度显著降低,罗伊氏乳杆菌丰度也显著降低。DCA饮水的INS-GAS小鼠胃体及胃窦部黏膜组织muc2 mRNA表达均显著高于NS饮水的INS-GAS小鼠(P均< 0.05)。此外,同为DCA饮水的INS-GAS小鼠胃内菌群丰度显著高于FVB/N小鼠(ACE指数P =0.022; Chao1指数P =0.028),两组小鼠组内差异大于组间差异,但尚无统计学意义(R=-0.056,P =0.647)。结论:DCA改变了INS-GAS 小鼠胃内菌群结构,主要为蓝藻细菌属和毛螺菌科Anaerostipes菌属相对丰度增高,乳杆菌属相对丰度降低,且促进了胃黏膜肠化标志物muc2表达。

    Abstract

    Objective:We aimed to investigate the effects of a secondary bile acid deoxycholic acid(DCA)on the gastric microbial community in INS-GAS(Insulin-Gastrin)mice model of intestinal type gastric cancer. Methods:Ten 8-week-old male INS-GAS mice were randomly divided into two groups,one group was given 0.2% DCA drinking water,and the other group was given normal saline (NS)solvent. Another 5 male FVB/N wild -type mice in the same litter were given 0.2% DCA as a genotype control group. After 3 months of drinking water intervention,the gastric contents of each group were collected for 16S rDNA amplicon sequencing,and the relative abundance of gastric microbiota,α diversity,β diversity and significantly different species were analyzed. The gastric body and antrum mucosal tissues were isolated respectively,and the mRNA expressions of intestinal markers cdx2,klf5,vil1 and muc2 were detected by qRT-PCR. Results:Compared with the NS group,DCA treatment changed the dominant bacteria in the stomachs of INS- GAS mice,and the bacteria with the highest relative abundance on the level of genus changed from the Lactobacillus(61.93%)to the undefined Cyanobacteria genus(58.70%). There were no remarkably differences in the abundance and diversity of gastric microflora between the two groups(ACE index P =0.213,Chaol index P =0.280;Shannon index P =0.391,Simpson index P =0.205). Principal component analysis found that the two groups had a considerable difference in the structure of the gastric microbiota,and the difference between the two groups was greater than the difference within the groups(R =0.368,P =0.032). DCA treatment significantly increased the abundance of Cyanobacteria in the stomach of INS -GAS mice at the levels of phylum,class,order,family and genus,and also upregulated the abundance of the Anaerostipes genus and the Cyanobacteria genus Lolium perenne and Phaseolus vulgaris. In addition, significantly higher enrichment of Lactobacillus including Lactobacillus reuteri,belonging to the phylum Firmicutes was found in gastric contents of INS -GAS mice drinking DCA compared with the control group. The expression of muc2 mRNA in the gastric body and antrum mucosa of INS-GAS mice drinking DCA was considerably higher than that of INS-GAS mice drinking NS(both P < 0.05). The abundance of gastric microbiota in the stomach of INS-GAS mice with the same DCA intervention was significantly higher than that of FVB/N mice(ACE index P =0.022;Chao1 index P =0.028). However,INS-GAS(DCA)and FVB/N(DCA)mice had greater differences within groups than between groups(R =-0.056,P =0.647). Conclusion:DCA intake changed the structure of the gastric microbiota of INS-GAS mice. The relative abundances of Cyanobacteria and Anaerossipes were significantly increased,while the relative abundance of Lactobacillus decreased,and promoted the expression of Muc2 mRNA.

  • 在消化道微生物研究史上,胃器官因其酸性环境曾被认为不适合细菌生长。1982 年 Marshall 和 Warren 发现胃内幽门螺杆菌(Helicobacter pylori, Hp)的存在及定植,推动了科学界对胃内菌群的认识。胃内菌群丰度介于1×102~1×104 CFU/g,并受到胃内酸碱度影响而动态变化[1]。通过对使用了下一代测序技术、16S rDNA一代测序技术、菌群指纹识别技术及 PhyloChip 芯片技术的 8 项研究汇总分析发现,人胃内菌群门水平丰度最高为变形菌门、厚壁菌门、拟杆菌门、放线菌门和梭杆菌门[2]。越来越多的证据表明,胃内菌群刺激炎症发生、促进细胞增殖、干细胞生理失调和异常代谢物产生,进而促进胃癌发生发展[3]。其中,Hp是公认的一类致癌因子[4],除Hp外其他胃内细菌也参与了致癌过程。16S rRNA基因测序发现胃癌患者胃黏膜乳杆菌属、轻型链球菌、副血链球菌、普氏菌属、韦荣球菌属丰度显著增高[5]

  • 早在1978年,临床研究就发现次级胆汁酸特别是脱氧胆酸(deoxycholic acid,DCA)是胃内十二指肠反流液的主要成分。疏水性次级胆汁酸通过促进DNA损伤、调节基因表达及胆汁酸受体、影响肠道菌群等方式促进多种消化道肿瘤(包括食管、胃、结肠及肝胆、胰腺)发生发展[6]。近期有研究采用液相色谱串联质谱法分析慢性浅表性胃炎、萎缩肠化及胃癌组织胃液中胆汁酸谱,发现胃液内DCA水平随患者胃黏膜异型程度而逐步升高[7]。基于FVB/N 近交系遗传背景的胰岛素⁃胃泌素(insulin⁃gastrin, INS⁃GAS)基因工程小鼠血清胃泌素过表达,促进胃腺体萎缩肠化、异型增生甚至癌变。20个月龄的INS⁃ GAS小鼠可自发形成胃粘膜萎缩肠化、异型增生、原位癌,甚至是伴有血管侵犯的进展期胃癌,而同月龄野生型FVB/N小鼠未见上述改变,因此INS⁃GAS小鼠是模拟人肠型胃癌发生发展的良好动物模型[8-9]

  • 16S rDNA 扩增子测序(16S rDNA amplicon se⁃ quencing)针对细菌核糖体小亚基16S rRNA的保守区域设计通用引物进行PCR扩增,对V3⁃V4高变区域进行测序分析和菌种鉴定,是研究机体菌群组成结构的重要手段。本研究旨在应用16S rDNA扩增子测序探究脱氧胆酸对肠型胃癌模型INS⁃GAS小鼠胃内菌群结构的影响,以期揭示这种胃内菌群变化对胃癌发生发展的潜在作用。

  • 1 对象和方法

  • 1.1 对象

  • FVB/N⁃Tg(Ins1⁃GAS)1Sbr/J 基因工程小鼠(简称INS⁃GAS小鼠)委托江苏集萃药康生物科技有限公司自美国 JAX 实验室引进并稳定繁育。本项研究共纳入10只7周龄雄性INS⁃GAS小鼠及5只同窝雄性FVB/N野生型小鼠,体重23~25 g[动物合格证号为SCXK(苏)2018⁃0008],于南京医科大学五台动物房无特定病原体(specific pathogen free,SPF)级别屏障设施饲养。5 只野生型 FVB/N 小鼠饲养在一笼,10只INS⁃GAS转基因小鼠随机平均分配到2笼,每笼5只。所有小鼠适应环境1周后达8周龄时开始正式实验,本研究通过南京医科大学实验动物福利伦理委员会审核批准(IACUC⁃2001024)。

  • 1.2 方法

  • 1.2.1 模型建立

  • 15只实验小鼠分为3组,分组如下:①5只FVB/N 野生型小鼠,饮用 0.2% DCA(实验组的基因型对照);②5只INS⁃GAS小鼠,饮用生理盐水(normal sa⁃ line,NS)(实验组的饮水干预对照);③5只INS⁃GAS 小鼠,饮用0.2% DCA(实验组)。0.2% DCA饮水浓度参考既往文献[10],脱氧胆酸钠购自美国Sigma⁃Al⁃ drich公司,生理盐水作为DCA的溶剂。所有小鼠自由进食采水,每周更换2次新鲜饮水,每日观察小鼠活动状态,每周观察小鼠进食量及称重。持续饮水干预3个月后,异氟烷麻醉后快速颈椎脱臼处死小鼠,游离鼠胃,沿胃大弯剖开取胃内容物。胃内容物标本在-80℃运送至苏州帕诺米克生物医药科技有限公司行16S rDNA扩增子测序。胃黏膜组织迅速放至冰PBS中漂洗除去残余胃内容物,然后按部位将黏膜分为胃体及胃窦,暂存于-80℃并尽快进行提 RNA、逆转录及荧光实时定量PCR测定。

  • 1.2.2 胃内菌群分析

  • 菌种相对丰度分析:将相似性超过97%的操作分类单元(operational taxonomic unit,OTU)聚类分析进行物种注释,分析每组样本在“门、纲、目、科、属、种”各分类水平丰度排名前10的菌种及其比例。

  • α多样性指数分析组间差异,反映各组样本胃内菌群多样性和丰富度。采用 ACE 指数和 Chao1 指数分析各组小鼠胃内菌群丰度,Shannon 指数和 Simpson指数分析菌群多样性。

  • β多样性分析是针对各个样本间菌群结构差异的比较,基于OTU水平的主成分分析(principal com⁃ ponent analysis,PCA)可以将这种菌落结构的差异进行二维展示,通过各个样本的聚散情况来判断各组菌群结构差异度。

  • 差异物种分析采用LEfSe(LDA Effect Size)分析各组具有统计学差异的物种(生物标志物),包括 LDA值分布柱状图、进化分支图和各组生物标志物丰度比较图,本研究纳入LDA值分布柱状图。

  • 1.2.3 胃体及胃窦组织肠化标志物 mRNA 表达水平测定

  • 使用TRIzol提取组织总RNA并测定浓度,采用日本 TaKaRa 公司 PrimeScriptTM RT Master Mix 试剂盒和TB Green Premix Ex Taq Ⅱ(RR820A)试剂盒依次进行逆转录及实时定量 PCR。所用小鼠引物序列(5′→3′)如下:①Gapdh正向引物AGGTCGGTGT⁃GAACGGATTTG,反向引物 GGGGTCGTTGATG⁃ GCAACA;② Cdx2 正向引物 CCGGAGACGATCT⁃ GAAACACG,反向引物 GTGATGGTGCGCGTGG⁃ TAT;③ Klf5 正向引物 CCGGAGACGATCTGAAA⁃ CACG,反向引物 GTTGATGCTGTAAGGTATGCCT; ④Vil1 正向引物 TCAAAGGCTCTCTCAACATCAC,反向引物 AGCAGTCACCATCGAAGAAGC;⑤Muc2 正向引物 ATGCCCACCTCCTCAAAGAC,反向引物 GTAGTTTCCGTTGGAACAGTGAA。扩增程序为阶段1:预变性95℃ 30 s;阶段2:PCR反应,共40个循环,每个循环95℃ 5 s,60℃ 30 s,95℃ 15 s;阶段3:跑融解曲线,60℃ 1 min,最后95℃ 15 s结束。2⁃ΔΔCT 法计算肠化标志物 cdx2、klf5、vil1 和 muc2 mRNA 相对表达水平。

  • 1.3 统计学方法

  • 本项研究实验组为 DCA 饮水干预 INS⁃GAS 小鼠,设置两个对照组,分别为NS饮水的INS⁃GAS小鼠 (饮水干预的对照组)和DCA饮水的FVB/N小鼠(基因型对照组),因此后续进行INS⁃GAS(NS)组与INS⁃ GAS(DCA)组、FVB/N(DCA)组与INS⁃GAS(DCA)组的两两比较。应用R软件进行统计分析,α多样性指数分析采用两组之间Wilcoxon秩和检验,β多样性分析采用非参数检验Anosim分析判断组间差异是否显著大于组内差异,LDA评分大于4为组间丰度有显著差异的菌种。P < 0.05为差异有统计学意义。

  • 2 结果

  • 2.1 各组小鼠一般情况

  • 在3个月的饮水干预期内,各组小鼠均无死亡,活跃度可,且进食量无显著差异(P > 0.05)。在此基础上,分析了各组小鼠实验期间体重变化,发现INS⁃ GAS(DCA)小鼠体重与其他两组相比,均无显著差异(P均> 0.05,图1)。

  • 2.2 各组小鼠胃内菌群分析

  • 2.2.1 各组小鼠胃内优势菌种分析

  • 图2A、B分别为门和属水平上各组小鼠胃内相对丰度排名前 10 的菌种。在门水平上,INS⁃GAS (NS)小鼠胃内相对丰度最高菌种为厚壁菌门 (66.84%),其次为蓝藻细菌门(29.72%);DCA 饮水改变了 INS⁃GAS 小鼠胃内优势菌种,蓝藻细菌门 (58.70%)占比高于厚壁菌门(34.06%);而在FVB/N (DCA)小鼠胃内两者相对丰度大致相同,分别为蓝藻细菌门(45.30%)、厚壁菌门(51.55%)。

  • 在属水平上,INS⁃GAS(NS)小鼠胃内相对丰度最高菌种为乳杆菌科菌属(61.93%),其次为未定义蓝藻细菌属(29.72%);DCA饮水改变了INS⁃GAS小鼠胃内优势菌种排序,未定义蓝藻细菌属(58.70%) 占比高于乳杆菌科菌属(30.41%);在FVB/N(DCA) 小鼠胃内两者相对丰度大致相同,分别为未定义蓝藻细菌属(45.30%)、乳杆菌科菌属(48.16%)。

  • 图1 各组小鼠实验期间体重变化

  • Figure1 Changes in body weight of mice in each group during the experiment

  • 2.2.2 各组小鼠胃内菌群丰度及多样性分析

  • 图3A、B分别为采用ACE指数和Chao1指数分析各组小鼠胃内菌群的丰度,结果发现 INS ⁃GAS (NS)和INS⁃GAS(DCA)两组小鼠胃内菌群丰度无显著差异(ACE指数P =0.213;Chao1指数P =0.280),而同为DCA饮水干预的INS⁃GAS(DCA)小鼠胃内菌群丰度显著高于FVB/N(DCA)小鼠(ACE指数P =0.022; Chao1指数P =0.028)。

  • 图3C、D为分别采用Shannon指数和Simpson指数分析各组小鼠胃内菌群多样性,结果发现DCA饮水干预不会影响 INS ⁃GAS 小鼠胃内菌群多样性 (Shannon指数P =0.391;Simpson指数P =0.205)。

  • 图2 各组小鼠胃内优势菌种分析

  • Figure2 Analysis of dominant bacteria in the stomachs of each group of mice

  • 2.2.3 各组小鼠胃内菌群结构差异度分析

  • 如图4A所示,PCA分析可见INS⁃GAS(DCA)小鼠与INS⁃GAS(NS)小鼠距离离散,说明两组小鼠胃内菌群差异性大。图4B、C为Anosim 法分析两组间差异和两组内差异相对大小,结果发现 INS ⁃GAS (DCA)和 INS ⁃ GAS(NS)两组小鼠 R 值 >0(R = 0.368),说明组间差异大于组内差异,且差异具有显著性(P =0.032);而INS⁃GAS(DCA)和FVB/N(DCA)两组小鼠R值<0(R =-0.056),说明组内差异大于组间差异,即基因型因素对两组小鼠胃内菌群影响大于DCA 饮水干预,但此种差异尚无统计学意义(P =0.647)。

  • 图3 各组小鼠胃内菌群丰度及多样性分析

  • Figure3 Analysis of the abundance and diversity of gastric microflora among the three groups

  • 图4 各组小鼠胃内菌群组间差异分析

  • Figure4 Analysis of the differences between groups of gastric microbiotas

  • 2.2.4 各组小鼠胃内菌群差异物种分析

  • 最后,分析了 INS ⁃GAS(NS)小鼠和 INS ⁃GAS (DCA)小鼠胃内各个水平上丰度具有显著差异的菌种(图5),旨在寻找在DCA干预下胃内潜在细菌标志物。结果发现,与NS对照组相比,DCA饮水干预促进蓝藻细菌在门、纲、目、科和属各个水平上显著增高,在种水平上也发现两种蓝藻细菌属Lolium pe⁃ rennePhaseolus vulgaris 丰度显著增高。此外, DCA饮水干预促进INS⁃GAS小鼠胃内毛螺菌科An⁃ aerostipes菌属丰度显著增高。

  • DCA饮水同样导致INS⁃GAS小鼠胃内某些细菌种类丰度显著降低,如厚壁菌门、杆菌纲和乳杆菌(目、科和属水平)丰度降低,在种水平上进一步发现了罗伊氏乳杆菌Lactobacillus reuteri丰度显著降低。

  • 2.3 胃体及胃窦组织肠化标志物 mRNA 相对表达水平分析

  • 鉴于既往文献报道DCA诱导胃上皮肠化生,进而诱导胃癌发生[711],采用荧光实时定量 PCR 实验 (qRT⁃PCR)测定各组小鼠胃体及胃窦黏膜组织肠化标志物mRNA表达水平(图6)。图6A、B分别为各组小鼠胃体及胃窦组织肠化标志物 cdx2、klf5、vil1 和 muc2 mRNA 相对表达水平,可见 DCA 饮水的 INS ⁃ GAS小鼠胃体及胃窦部黏膜组织muc2 mRNA表达均显著高于NS饮水的INS⁃GAS小鼠(均P < 0.05),表明 DCA诱导INS⁃GAS小鼠胃黏膜肠化发生。

  • 3 讨论

  • 近年来胃内菌群逐渐引起关注,但非 Hp 的胃内其他细菌研究较少,在胃癌发生发展中的作用尚不清楚。毛螺菌科 Anaerostipes 菌属是人体肠道内主要的产丁酸菌之一[12]。有研究将 AnaerostipescaccaeA. caccae)JCM 13 470株和D10株分别在添加初级胆汁酸的培养基中培养24 h,发现A. caccae具有将初级胆汁酸转化为相应次级胆汁酸的能力[13]。本研究发现Anaerostipes菌属丰度在DCA饮水干预INS ⁃GAS小鼠胃内显著升高,表明Anaerostipes菌属可能参与了胃内胆汁酸代谢谱调节。

  • 图5 INS⁃GAS(DCA)与INS⁃GAS(NS)两组小鼠胃内丰度具有显著差异的菌种

  • Figure5 Significantly different species in the stomachs of the INS⁃GAS(DCA)and INS⁃GAS(NS)groups

  • 图6 各组小鼠胃体及胃窦黏膜组织肠化标志物mRNA表达水平

  • Figure6 mRNA expression levels of intestinal metaplastic markers in gastric body and antrum mucosa of mice in each group

  • 现有研究表明,蓝藻细菌在胃内可能发挥有益作用,如蓝藻细菌在Hp阴性慢性浅表性胃炎患者、胃上皮肠化生患者及胃癌患者胃内丰度逐步减低,差异具有统计学意义(14.0% vs 4.2% vs. 0.04%,P < 0.001),且Hp感染患者根除治疗后胃内蓝藻细菌丰度也不会恢复[14]。另有研究发现蓝藻细菌提取物多糖成分可以抑制Hp向胃黏膜附着[15]。本研究发现DCA饮水干预后,INS⁃GAS小鼠胃内蓝藻细菌在门、纲、目、科、属水平甚至某些蓝藻菌种丰度显著升高,表明 DCA 对胃内菌群可能存在某种有益调节。由于相关文献甚少,因此需要进一步明确胃内胆汁酸水平与蓝藻细菌丰度的关系及意义。

  • 乳杆菌属在所有哺乳动物胃内均存在,可以定植于人胃黏膜上。胃内乳杆菌属可将乳糖转化为乳酸从而促使胃黏膜表面酸化,因此能够适应胃内酸性环境并定植。目前胃内乳杆菌属对胃黏膜影响存在两种矛盾观点及相应依据。第一种观点认为,Hp感染导致壁细胞损伤,胃内pH值上升,而乳杆菌属可以分泌乳酸,降低胃内 pH 值。据文献报道,约氏乳杆菌Lactobacillus johnsonii、鼠乳杆菌Lac⁃ tobacillus murinus 及罗伊氏乳杆菌在体外实验中可以抑制Hp的生长、黏附及定植[16],因此认为胃内乳杆菌属拮抗了Hp对胃黏膜的有害作用。另一些研究发现,胃癌患者胃黏膜乳杆菌属丰度较正常人升高,其中Lactobacillus coleohominis丰度在胃炎、胃化生及胃癌患者胃内逐步升高,表现出潜在的促癌特性[17],这可能是由于乳杆菌属产生乳酸,为异型增生组织及血管生长提供能量。本研究中,与 INS⁃ GAS(NS)小鼠相比,在不同水平发现 INS ⁃ GAS (DCA)小鼠胃内厚壁菌门、杆菌纲、乳杆菌目、乳杆菌科、乳杆菌属及罗伊氏乳杆菌丰度显著降低。从属水平上看,INS⁃GAS(NS)小鼠胃内乳杆菌科菌属相对丰度占比61.93%,而在INS⁃GAS(DCA)组小鼠仅为30.41%。其中,乳杆菌属归属于厚壁菌门杆菌纲,因此INS⁃GAS(DCA)小鼠胃内菌群门水平上厚壁菌门、纲水平上杆菌纲丰度下降可能是由于乳杆菌在胃内丰度大幅度降低引起。

  • 本研究尚存在一些局限性,如本研究取小鼠胃内容物进行菌群测定,而没有取胃黏膜。既往针对人胃内菌群进行的测定研究,有的采用胃黏膜样本[18],有的采用胃液样本[7]。本研究通过多次解剖发现,小鼠胃内不存在胃液,而仅存在胃内容物。此外,也有动物研究采用胃内容物样本进行胃内菌群测定[19]。因此,本研究测定的小鼠胃内容物中菌群成分,可以视为类似于人胃液内菌群结构测定。本研究在实验分组上存在一些不完善之处,在不考虑小鼠数量限制的前提下设置4个分组更为严谨,即 FVB/N(NS)、FVB/N(DCA)、INS⁃GAS(NS)和 INS⁃GAS(DCA)4个干预组。另外,仅采用DCA饮水干预3个月,后续应进一步延长干预时间至INS⁃GAS 小鼠成瘤,明确小鼠胃癌发生时胃内菌群变化。

  • 综上所述,次级胆汁酸虽是除 Hp 外的另一重要致胃癌因子,但本研究发现DCA饮水干预尚存在对胃内菌群潜在的有益调节,如蓝藻细菌属丰度增加,促癌乳杆菌属丰度降低。毛螺菌科Anaerostipes 菌属虽是肠道产丁酸菌属,但在胃内作用尚不清楚,既往文献尚未见报道在胃癌发生发展中丰度改变的情况。本研究发现 DCA 干预后 Anaerostipes菌属丰度显著上升,此种变化仍需后续深入探究。

  • 参考文献

    • [1] NARDONE G,COMPARE D.The human gastric microbi⁃ ota:Is it time to rethink the pathogenesis of stomach dis⁃ eases[J].United European Gastroenterol J,2015,3(3):255-260

    • [2] HUNT R H,YAGHOOBI M.The esophageal and gastric microbiome in health and disease[J].Gastroenterol Clin North Am,2017,46(1):121-141

    • [3] ABREU M T,PEEK R M.Gastrointestinal malignancy and the microbiome[J].Gastroenterology,2014,146(6):1534-1546.e3

    • [4] 周铖,孙鹏飞,解昕轶,等.质子泵抑制剂对消化道菌群组成变化的相关性研究进展[J].南京医科大学学报(自然科学版),2020,40(9):1386-1390

    • [5] DICKSVED J,LINDBERG M,ROSENQUIST M,et al.Mo⁃ lecular characterization of the stomach microbiota in pa⁃ tients with gastric cancer and in controls[J].J Med Micro⁃ biol,2009,58(4):509-516

    • [6] DI CIAULA A,WANG D Q,MOLINA⁃MOLINA E,et al.Bile acids and cancer:direct and environmental ⁃ depen⁃ dent effects[J].Ann Hepatol,2017,16(Suppl.1:s3 ⁃ 105.):s87-s105

    • [7] LEE W,UM J,HWANG B,et al.Assessing the progres⁃ sion of gastric cancer via profiling of histamine,histidine,and bile acids in gastric juice using LC⁃MS/MS[J].J Ste⁃ roid Biochem Mol Biol,2020,197:105539

    • [8] QUINN L,SHEH A,ELLIS J L,et al.Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS⁃GAS mice triggers dysbiosis and vitamin K de⁃ ficiency resulting in gastric hemorrhage[J].Gut Mi⁃ crobes,2020,11(4):820-841

    • [9] PINZON⁃GUZMAN C,MEYER A R,WISE R,et al.Eval⁃ uation of lineage changes in the gastric mucosa following infection with helicobacter pylori and specified intestinal flora in INS⁃GAS mice[J].J Histochem Cytochem,2019,67(1):53-63

    • [10] QUANTE M,BHAGAT G,ABRAMS J A,et al.Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett ⁃like metaplasia[J].Cancer Cell,2012,21(1):36-51

    • [11] YUAN T,NI Z,HAN C,et al.SOX2 interferes with the function of CDX2 in bile acid ⁃induced gastric intestinal metaplasia[J].Cancer Cell Int,2019,19:24

    • [12] RIVIÈRE A,SELAK M,LANTIN D,et al.Bifidobacteria and butyrate ⁃ producing colon bacteria:importance and strategies for their stimulation in the human gut[J].Front Microbiol,2016,7:979

    • [13] TAWTHEP S,FUKIYA S,LEE J Y,et al.Isolation of six novel 7⁃oxo ⁃ or urso ⁃type secondary bile acid ⁃producing bacteria from rat cecal contents[J].J Biosci Bioeng,2017,124(5):514-522

    • [14] PARK C H,LEE A R,LEE Y R,et al.Evaluation of gas⁃ tric microbiome and metagenomic function in patients with intestinal metaplasia using 16S rRNA gene sequenc⁃ ing[J].Helicobacter,2019,24(1):e12547

    • [15] LOKE M F,LUI S Y,NG B L,et al.Antiadhesive proper⁃ ty of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin[J].FEMS Immunol Med Microbiol,2007,50(2):231-238

    • [16] ZAMAN C,OSAKI T,HANAWA T,et al.Analysis of the microbial ecology between Helicobacter pylori and the gastric microbiota of mongolian gerbil[J]s.J Med Micro⁃ biol,2014,63(Pt 1):129-137

    • [17] AVILES⁃JIMENEZ F,VAZQUEZ⁃JIMENEZ F,MEDRA⁃ NO ⁃GUZMAN R,et al.Stomach microbiota composition varies between patients with non⁃atrophic gastritis and pa⁃ tients with intestinal type of gastric cancer[J].Sci Rep,2014,4:4202

    • [18] FERREIRA R M,PEREIRA ⁃ MARQUES J,PINTO ⁃ RI⁃ BEIRO I,et al.Gastric microbial community profiling re⁃ veals a dysbiotic cancer ⁃ associated microbiota[J].Gut,2018,67(2):226-236

    • [19] KANG X,ZHAN L,LU X,et al.Characteristics of gastric microbiota in GK rats with spontaneous diabetes:a compar⁃ ative study[J].Diabetes Metab Syndr Obes,2020,13:1435-1447

  • 参考文献

    • [1] NARDONE G,COMPARE D.The human gastric microbi⁃ ota:Is it time to rethink the pathogenesis of stomach dis⁃ eases[J].United European Gastroenterol J,2015,3(3):255-260

    • [2] HUNT R H,YAGHOOBI M.The esophageal and gastric microbiome in health and disease[J].Gastroenterol Clin North Am,2017,46(1):121-141

    • [3] ABREU M T,PEEK R M.Gastrointestinal malignancy and the microbiome[J].Gastroenterology,2014,146(6):1534-1546.e3

    • [4] 周铖,孙鹏飞,解昕轶,等.质子泵抑制剂对消化道菌群组成变化的相关性研究进展[J].南京医科大学学报(自然科学版),2020,40(9):1386-1390

    • [5] DICKSVED J,LINDBERG M,ROSENQUIST M,et al.Mo⁃ lecular characterization of the stomach microbiota in pa⁃ tients with gastric cancer and in controls[J].J Med Micro⁃ biol,2009,58(4):509-516

    • [6] DI CIAULA A,WANG D Q,MOLINA⁃MOLINA E,et al.Bile acids and cancer:direct and environmental ⁃ depen⁃ dent effects[J].Ann Hepatol,2017,16(Suppl.1:s3 ⁃ 105.):s87-s105

    • [7] LEE W,UM J,HWANG B,et al.Assessing the progres⁃ sion of gastric cancer via profiling of histamine,histidine,and bile acids in gastric juice using LC⁃MS/MS[J].J Ste⁃ roid Biochem Mol Biol,2020,197:105539

    • [8] QUINN L,SHEH A,ELLIS J L,et al.Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS⁃GAS mice triggers dysbiosis and vitamin K de⁃ ficiency resulting in gastric hemorrhage[J].Gut Mi⁃ crobes,2020,11(4):820-841

    • [9] PINZON⁃GUZMAN C,MEYER A R,WISE R,et al.Eval⁃ uation of lineage changes in the gastric mucosa following infection with helicobacter pylori and specified intestinal flora in INS⁃GAS mice[J].J Histochem Cytochem,2019,67(1):53-63

    • [10] QUANTE M,BHAGAT G,ABRAMS J A,et al.Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett ⁃like metaplasia[J].Cancer Cell,2012,21(1):36-51

    • [11] YUAN T,NI Z,HAN C,et al.SOX2 interferes with the function of CDX2 in bile acid ⁃induced gastric intestinal metaplasia[J].Cancer Cell Int,2019,19:24

    • [12] RIVIÈRE A,SELAK M,LANTIN D,et al.Bifidobacteria and butyrate ⁃ producing colon bacteria:importance and strategies for their stimulation in the human gut[J].Front Microbiol,2016,7:979

    • [13] TAWTHEP S,FUKIYA S,LEE J Y,et al.Isolation of six novel 7⁃oxo ⁃ or urso ⁃type secondary bile acid ⁃producing bacteria from rat cecal contents[J].J Biosci Bioeng,2017,124(5):514-522

    • [14] PARK C H,LEE A R,LEE Y R,et al.Evaluation of gas⁃ tric microbiome and metagenomic function in patients with intestinal metaplasia using 16S rRNA gene sequenc⁃ ing[J].Helicobacter,2019,24(1):e12547

    • [15] LOKE M F,LUI S Y,NG B L,et al.Antiadhesive proper⁃ ty of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin[J].FEMS Immunol Med Microbiol,2007,50(2):231-238

    • [16] ZAMAN C,OSAKI T,HANAWA T,et al.Analysis of the microbial ecology between Helicobacter pylori and the gastric microbiota of mongolian gerbil[J]s.J Med Micro⁃ biol,2014,63(Pt 1):129-137

    • [17] AVILES⁃JIMENEZ F,VAZQUEZ⁃JIMENEZ F,MEDRA⁃ NO ⁃GUZMAN R,et al.Stomach microbiota composition varies between patients with non⁃atrophic gastritis and pa⁃ tients with intestinal type of gastric cancer[J].Sci Rep,2014,4:4202

    • [18] FERREIRA R M,PEREIRA ⁃ MARQUES J,PINTO ⁃ RI⁃ BEIRO I,et al.Gastric microbial community profiling re⁃ veals a dysbiotic cancer ⁃ associated microbiota[J].Gut,2018,67(2):226-236

    • [19] KANG X,ZHAN L,LU X,et al.Characteristics of gastric microbiota in GK rats with spontaneous diabetes:a compar⁃ ative study[J].Diabetes Metab Syndr Obes,2020,13:1435-1447

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