The Influence Of Vaginal Microbiome And Metabolites On HPV Infecting Human Epithelial Cells

Cervical cancer is one of the most common tumors threatening the life and health of women all over the world.It is also one of the few malignant tumors with clear etiology.Currently,it has been confirmed that the continuous infection of high-risk HPV is a necessary condition for the occurrence and development of cervical cancer prelesions and cervical cancer.80%of women infected with HPV will be cleared within 1 year,only a small number of women after infection with persistent infection or even cervical lesions.The mechanism by which the body removes persistent HPV infection is unclear.Vaginal microecology mainly includes vaginal microbiome,endocrine regulation and vaginal anatomy.The vaginal microbiome,as a biological barrier,plays an important role in maintaining the acidic environment of the vagina and activating the immune function.With the rapid development of macrogenomics,macrotranscriptome,proteomics,metabolomics and so on,the study of vaginal microbiome,which is closely related to the health of female reproductive tract,is also deepening.The vaginal microbiome of healthy Asian women is dominated by lactobacillus lactobacillus,which maintains the dynamic balance of the reproductive tract with the participation of sex hormones(estrogen,progesterone,etc.).When the system is out of balance,it will increase the risk of pathogen infection(mold,trichomonas,mycoplasma,chlamydia,neisseria gonorrhoeae,HPV,HIV,etc.)and cervical diseases,but the relationship between vaginal flora and HPV infection is still not very clear.In this study,16S rRNA gene sequencing was first performed on vaginal secretion samples.On this basis,peptidoglycan,a cell wall component of lactobacillus,was used to observe the expression of HSPG,the main membrane receptor of HPV in human epithelial cells(HaCat),and the changes of HPV pseudovirus granule infection of cells.Objective to analyze the effect of vaginal microbiome and its metabolites on human epithelial cells infection of HPV,and to provide evidence for the prevention of HPV infection and cervical cancer.Part ? The analysis on the relationship between hr-hpv infection and the composition and diversity of vaginal microbiomeObjective:To explore the relationship between the community composition and diversity ofvaginal microbiome and hr-hpv infection.Methods:In the Colposcopy room of Women's Hospital,Zhejiang University,samples of HPV negative and HPV positive patients were collected according to the proposed na pai standard.After the DNA of specimens was extracted,66 samples which meeting the requirements of database construction were selected and sent to the company for,and corresponding results were obtained.According to the corresponding sequencing results,the relationship between vaginal microbiome and hr-hpv infection was analyzed.Results:1.Through 16S rRNA gene sequencing,lactobacillus was found to occupy a large proportion in the HPV negative and HPV positive groups,which was the dominant bacteria genus.At the genus level,there was no significant difference between the two groups.2.The diversity of vaginal microbiomeof HPV positive group was decreased.Conclusions:The diversity of vaginal microbiome in HPV positive women was lower at the generic level than in HPV negative women.In the HPV positive state,the proportion of firmicutes,lactobacillus and so onincreased.Part ? Effect of peptidoglycan from L.crispatus cell wall's component on HPV infecting human epithelial cellsObjective:To determine the effect of lactobacillus metabolite peptidoglycan(PGN)on HPV infecting epithelial cells.Methods:According to the results of the sequencing analysis,anchoring L.crispatus and its relevant metabolites peptidoglycan(PGN)to do the next step.After the extraction and verification of PGN material,Human epithelial cells(HaCat)were treated with peptidoglycan for 24h to construct a cell model of pgn-hacat cells infected with HPV pseudovirus particles(PsV).Use RT-PCR to determine the expression of HSPG2,which is the main cell membrane receptor of HPV infecting cells.Use flow cytometry to determine the infection rate of PsV,which is marked with GFP fluorescence label.Results:1.The expression level of HSPG2 in HaCat cells affected by PGN was lower than that in the negative group.2.The PsV infection rate of HaCat cells decreased after PGN treatment compared with the negative group.Conclusion1.Certain concentration of peptidoglycan can inhibit the expression of HSPG2,the major cell membrane receptor related to HPV.2.The HPV infection rate of epithelial cells was reduced after the action of certain concentration of peptidoglycan.