| Before the discovery of Helicobacter pylori(Hp)in 1982,the stomach was thought to be sterile,and until the advent of high-throughput sequencing,research methods in the field of microbiology were more based on classical bacteriology.However,with the development of sequencing technology,people have realized the real existence and complexity of gastric flora.Among many technologies,ampli sequencing is an epoch-making product of method innovation.Combined with the high speed,high throughput and low cost of the next generation sequencing(NGS)technology,it is widely used in the detection and research of microorganisms in various fields.Different from the rapid development of intestinal flora,research on gastric flora is still in its infancy.Previous studies have shown that mucosal flora in advanced gastric cancer is dysbiosis.However,at the level of flora,there are no significant differences among non-atrophic gastric mucosa and atrophic gastric mucosa and intestinal metaplasia mucosa in stomach of non-neoplasia lesion(NNL),which are different from the cascadal progression of gastric cancer.Meanwhile,with the exception of Hp,the understanding of microbial environmental biology of early gastric cancer(EGC)is very limited.Combined with the concept of EGC recognition in the field of digestive endoscopy,this study focused on the background mucosa and attempted to explore the background mucosal microenvironment of different lesions from the perspective of microorganisms.Discussion on fundic gland polyp(FGP),chronic gastritis(CG),low-grade intraepithelial neoplasia(LGIN)and EGC gastric flora composition and changes are made.Mucosal microenvironment behind gastric mucosal neoplasia lesion(NL)and NNL were revealed from the perspective of microflora,so as to provide reference for clinical diagnosis and treatment decisions and classification of lesions from different perspectives.From July 2018 to June 2020,we sequenced and analyzed 252 gastric mucosa biopsies from 63 patients who participated in the study,and all patients were divided into four subgroups: Fundic Gland Polyp(FGP),Chronic Gastritis(CG),Low Grade Intraepithelial Neoplasia(LGIN),Early Gastric Cancer(EGC),among which the EGC group contains High Grade Intraepithelial Neoplasia(HGIN),which conforms to the pathological standards of gastric cancer in Japan.Samples from the FGP and CG groups were taken from normal gastric corpus and gastric antral,2 biopies each,a total of 4 biopsies.The samples of LGIN and EGC group were taken from the adjacent mucosa at the four quadrant of the endoscopic submucosal dissection horizontal margin.A total of 4biopsies were taken.By sequencing and analysis,we found the abundance and diversity of the microenvironment in the stomach,Helicobacter usually restricts the richness of other bacterial genera with absolute abundance advantage,and the microflora composed of Proteobacteria,Firmicutes,Bacteroidetes,Helicobacter,Prevotella,Streptococcus and other dominant species form an interweaving and complicated microenvironment network.In addition,we have also achieved the following innovative research results based on experiments and bioinformatics analysis:1.From the perspective of mucosal microenvironment,the microenvironment of NL(LGIN/EGC),FGP and CG is distinct from each other by showing significant differences in Alpha diversity and Beta diversity.Differential genus corresponding to Paracoccus,Blautia,Barnesiella,Lactobacillus,Thauera,Collinsella were identified in neoplasia lesions,while Pseudomonas and Kingella were depleted(p<0.001,fdr<0.01).LGIN and EGC showed no significant difference in microbiome diversity or composition,and there was positive interaction between them.In the prediction of gene function and metabolic pathway constituted by microbiota,they also maintain a high degree of consistency in the expression of pathway.This suggests the importance of judging NL as a group from the perspective of microenvironment.The above results demonstrate the importance of taking LGIN and EGC as the overall judgment of NL from the perspective of microenvironment,which may suggest that the confusion in the judgment of the grade of gastric intraepithelial neoplasia should be weakened in clinical practice,so as to conduct active follow-up and intervention for neoplastic lesions.In addition,FGP has its own unique microflora environment with high diversity and unique composition and structure,and has a unique flora network system to form resistance to Helicobacter.These findings based on the microenvironment point of view suggest that the microenvironment of microflora can be used as a potential biomarker for the identification of NL to serve clinical or scientific research.Age or gender factors are also correlated with the flora structure in terms of flora composition.Male or aging often has a positive correlation with the representative flora of the neoplastic lesion group,while female often has a positive correlation with the representative flora of the FGP group.This also reveals the prevalence trend of gastric cancer in terms of age and gender from the perspective of flora microenvironment.2.Based on the double-edged sword effect brought by the high sensitivity of next-generation sequencing,we added 16 S sequencing endoscopic quality control to minimize the pollution in tumor microenvironment research.In the numerous studies on mucosal flora based on sequencing technology,people seem to have neglected the potential bacteria-carrying ability of endoscopy as a tool for acquiring digestive tract mucosa.We extracted the aldehyde neutralizer eluent from the endoscopic biopsy pipeline through the filtration membrane and conducted 16 S sequencing.The results showed that bacteria could be detected in the endoscopic biopsy pipeline with a long service life,including Brevundimonas,which can produce spores,and Corynebacterium,Pseudomonas,which contain high content.Although the limitations of 16 S sequencing could not indicate the activity or not of the bacteria we measured,for the rigor of the study,we followed the results of quality control based on 16 S sequencing endoscopy and judged that the aged endoscopy was not suitable for mucosal biopsy during 16 S sequencing.This problem,which is easy to be ignored by people,reminds us that the quality control of endoscopy is very critical in both clinical and scientific research tasks. |
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