Characteristics Of Vaginal Flora And The Study On The Mechanism Of Differential Bacterial Biofilm Inducing Cervical Canceration

[Objective]High risk human papillomavirus(HR-HPV)infection is the cause of cervical cancer,but not all infected people develop cervical cancer.In most cases,the virus is cleared by the immune system,and only persistent infection can develop cervical cancer.The body's ability to clear HPV is highly susceptible to vaginal microecology.The imbalance of vaginal flora is the main synergistic factor for HR-HPV to infect the cervix for a long time and eventually lead to cancer.In this study,the vaginal flora was detected at different stages from HR-HPV infection in the cervix to the development of cervical cancer.Obtain total flora information,study the relationship between changes in vaginal flora and HR-HPV infection and cervical cancer.The aim is to elucidate the change characteristics of the vaginal flora in the occurrence and development of cervical cancer,and to find the differential bacteria for further study.To seek new ideas for the prevention and treatment of cervical cancer.[Methods]We make a collection of 90 cases of female vaginal secretions swabs from The Third Affiliated Hospital of Kunming Medical University,from December 2017 to December 2018.30 cases of simple HR-HPV infection,30 patients with HR-HPV and diagnosed with cervical intraepithelial neoplasia(CIN),and 30 patients with HR-HPV infection were diagnosed as cervical squamous cell carcinoma Ibi.At the same time,30 healthy women with normal physical examination in the gynecological clinic were selected during the same period,and all of them were negative for HR-HPV.The total DNA of vaginal secretions was extracted,the qualified DNA samples were sent to high-throughput sequencing,and the sequencing results were analyzed by bioinformatics and statistical processing.The structural differences and changes of the vaginal flora were analyzed from the aspects of HR-HPV infection status and cervical lesion degree,and the most likely differential bacteria were finally selected for further research.[Results]? Lactobacillus is the absolute dominant flora in the vaginal flora of normal healthy women and HR-HPV infected patients(Richness>50%).Lactobacillus decreased in the level of phylum,class,order and family in HR-HPV infected patients,but there was no statistical difference(P>0.05);There were significant differences between the two groups in the distribution of microflora at six levels:phylum,class,order,family,genus and species(P<0.05);? Diversity of flora:The chao and ace index of healthy women and HR-HPV(+)women were the same,with no statistically significant diffrence(P>0.05),indicating that the total number of bacteria in the samples of the two groups was the same.The shannon index of HR-HPV(+)women was significantly higher,and the simpson index was significantly lower than that of healthy women(P<0.05).Those indicates that the structure and composition of HR-HPV(+)female vaginal flora have obvious diversity,complexity and heterogeneity;? Lactobacillus was the dominant bacteria in the vaginal flora of HR-HPV(+)female,cervical precancerous lesions and cervical cancer patients(Richness>50%).With the progress of the disease,Lactobacillus abundance decreased,but no statistical difference(P>0.05).The composition of the three groups of samples was significantly different at the six levels of phylum,class,order,family,genus and species,and the difference was statistically significant(P<0.05);? Diversity of flora:There was no statistically difference in the chao and ace index between HR-HPV(+)alone,cervical precancerous lesions,and cervical cancer patients(P>0.05),indicating that the richness of vaginal flora of the three groups of samples was similar.The difference of shannon and simpson index have statistically difference(P<0.05),indicating that the diversity of the three groups of samples was different.Comparison within the group found that:There was no significant difference in richness(chao index,ace index)or diversity(shannon index,simpson index)between the HR-HPV(+)infection group and the cervical precancerous group(P>0.05).These two groups were similar in structure and could be classified as non-cancer group.Compared with HR-HPV(+)group and cervical precancerous lesions group,cervical cancer group has more obvious diversity(shannon index,simpson index)of flora(P<0.05).And with the progress of the disease,the difference was more significant(P<0.05);? Differential analysis found that Gardnerella vaginalis was the main differential bacterium in the vaginal flora of patients with HR-HPV infection and cervical cancer(P<0.05);[Conclusion]Lactobacillus is the absolute dominant flora in women's vagina.Changes in the structure of the vaginal flora,especially the increased diversity and complexity of the flora,are related to HR-HPV infection and cervical cancer.Gardnerella vaginalis is the main differential bacterium in the vaginal flora of patients with HR-HPV infection and cervical cancer,and is the main synergistic factor for HR-HPV infection of the cervix and induction of cancer.[Objective]The first part of the study found that Gardnerella vaginalis was the main differential bacterium in the vaginal flora of patients with HR-HPV infection and cervical cancer.A large number of studies have shown that Gardnerella vaginalis has a strong ability to form biofilms,and mainly exists in the form of biofilms in female vagina.This part of the study intends to establish a model of Gardnerella vaginalis biofilm in vitro,and to describe its film formation rules,spatial structure and characteristics of biofilm.At the same time,the effects of estrogen levels that regularly change with women's menstrual cycles on the formation of Gardnerella vaginalis biofilms were explored.The purpose is to clarify the biofilm formation characteristics of Gardnerella vaginalis and establish the foundation for the successful extraction of mature biofilms in the next step.At the same time,the effect of estrogen on the formation of Gardnerella vaginalis biofilm was explored,and a new way for clinical treatment of Gardnerella vaginalis infection was found.[Methods]First,a model of Gardnerella vaginalis biofilm was established in vitro using a 96-well plate as a carrier.The amount of biofilm formation was measured by crystal violet semi-quantitative method to describe the biofilm formation law.Secondly,to simulate the estradiol concentration in women,culture the Gardnerella vaginalis biofilm in vitro with a medium containing different concentrations of estradiol(100pmol/L,200pmol/L,400pmol/L,1000pmol/L),and crystal violet The effect of estradiol at different concentrations on the formation of Gardnerella vaginalis biofilms was measured by a semi-quantitative method.Then,a model of Gardnerella vaginalis biofilm was established in vitro using a metal titanium sheet as a carrier,and its spatial structure and characteristics were observed under a scanning electron microscope,and the effects of different concentrations of estradiol on its spatial structure and characteristics were observed.The effects of estradiol at different concentrations on the thickness of Gardnerella vaginalis biofilm formation and the ratio of live/dead bacteria were measured by confocal laser.[Results]? Gardnerella vaginalis begins to gradually form a biofilm at 12h,enters a rapid growth period at 24h,and the biofilm production reaches a peak at 96h(P>0.05),and then does not increase again(P<0.05);? At 96h,the Gardnerella vaginalis biofilm exhibited a typical three-dimensional three-dimensional structure,showing a "mushroom cloud"-like stack,with a large number of channels and extracellular matrix in the middle;? Different concentrations of estradiol have an inhibitory effect on the formation of Gardnerella vaginalis biofilms(P<0.05);As the estradiol concentration increases(100pmol/L,200pmol/L,400pmol/L,1000pmol/L),the inhibitory effect gradually increases(P<0.05).400pmol/L is equivalent to 1000pmol/L of estradiol concentration,so 400pmol/L is the optimal concentration to inhibit the formation of Gardnerella vaginalis biofilm;? Observation under the laser confocal microscope found that the biofilm thickness of Gardnerella vaginalis gradually became thinner with the increase of estradiol concentration at the same time point(P<0.05).At the same concentration,the Gardnerella vaginalis biofilm gradually thickened over time(P<0.05);? Observed under scanning electron microscope and laser confocal microscope,it was found that at the same time point,the biofilm of the 100pmol/L group was mature than that of the 200pmol/L group(P<0.05),and the biofilm of the 200pmol/L group was mature than the 400pmol/L group(P<0.05);[Conclusion]Gardnerella vaginalis can form a dense biofilm on the surface of polyethylene and metal titanium,but the biofilm has a slower growth rate and a longer film formation time.Estradiol can inhibit the formation of Gardnerella vaginalis biofilm,and the inhibitory effect is related to the effect time and concentration.Estradiol mainly works by inhibiting the formation of Gardnerella vaginalis,delaying the formation of biofilm,and affecting the internal structure of biofilm.The appropriate concentration of estradiol can be used as the drug of choice for the treatment of Gardnerella biofilm infection in the vagina.[Objective]In the second part of the experiment,a model of Gardnerella vaginalis biofilm has been successfully established in vitro,which confirms that estradiol has an inhibitory effect on the formation of Gardnerella vaginalis biofilm.This part of the study intends to extract a pure biofilm based on the successful establishment of an in vitro model of Gardnerella vaginalis biofilm.Co-culture of Gardnerella vaginalis biofilm and cervical precancerous cells(Hs cells)to investigate the effect of Gardnerella vaginalis biofilm on the cellular function and epithelium mesenchymal transformation(EMT)of precervical carcinoma.The purpose is to discuss the molecular mechanism of Gardnerella vaginalis biofilm to induce cervical cancer.At the same time,the effects of different concentrations of estrogen on cervical precancerous cell epithelial-mesenchymal transition were studied to provide a basis for further evaluating the safety of estrogen in the treatment of Gardnerella biofilm infection in the vagina.[Methods]First,the Gardnerella vaginalis biofilm was successfully cultured in vitro and a pure bacterial membrane was extracted.Hs cells were cultured in vitro with media containing Gardnerella vaginalis biofilms with different concentrations.CCK8 was used to detect the proliferation of Hs cells and Transwell method was used to detect the migration of Hs cells.RT-PCR and Western-blot methods were used to detect the expression of epithelial marker E-cadherin,interstitial markers N-cadherin,Snail and Vimentin under different concentrations of biofilm stimulation.Estradiol(Opmol/L,200pmol/L,400pmol/L,800pmol/L)of different concentrations were co-cultured with H8 cells,and the expression of epithelial marker E-cadherin and interstitial marker Vimentin of H8 cells were detected by RT-PCR.[Results]? The proliferation of Hs cell can be inhibited by Gardnerella vaginalis biofilm(P<0.05),but Gardnerella vaginalis biofilm has no effect on Hs cell migration(P>0.05);? Gardnerella vaginalis biofilm induces low expression of epithelial marker(E-cadherin)and high expression of interstitial markers(N-cadherin,Snail,Vimentin)on Hs cells at the gene level(P<0.05);? Gardnerella vaginalis biofilm induced low expression of E-cadherin and high expression of interstitial marker proteins(N-cadherin,Snail,Vimentin)on H8 cells at the protein level;? Estradiol at different concentrations has no effect on the expression of EMT-related markers in H8 cells(P>0.05);[Conclusion]Gardnerella vaginalis biofilm has cytotoxic effects on cervical precancerous cells.Gardnerella vaginalis biofilms play a pro-cancer role by inducing epithelial mesenchymal transition in precervical carcinoma.Estradiol does not induce epithelial-mesenchymal transition in cervical precancerous cells,and it is safe to treat Gardnerella biofilm infection in the vagina.[Objective]The third part of the study found that Gardnerella vaginalis biofilm play a role in promoting cancer by inducing EMT in cervical precancerous cells.EMT has been recognized by academics as a key step in inducing cell canceration,but its mechanism and signal regulation in the Gardnerella vaginalis biofilm to promote cervical cancerization are not clear.In this part of the experiment,based on a previously established model of Gardnerella vaginalis biofilm-induced H8 cells,further study the detailed signal transduction process of Gardnerella vaginalis biofilm-induced EMT.The aim is to provide scientific basis for the biofilm of gardnerella vaginalis as a new target for clinical prevention and treatment of cervical cancer.[Methods]First,the indirect cellular immunofluorescence method was used to detect the expression of TLR4 and P-Smad2/3 protein in H8 cells after the action of Gardnerella vaginalis biofilm.To investigate the change trend of above factors in EMT,which induced by Gardner biofilm in H8 cells of vagina.The RT-PCR and Western-blot methods were used to study the regulatory mechanism of TGF-?1/Smad signaling pathway in Hscells EMT induced by Gardnerella vaginalis biofilm;Furthermore,by inhibiting the expression of TLR4 protein,the regulation of TLR4 on the signaling pathway of EMT and TGF-?1/Smad in H8 cells was investigated.[Results]? Immunofluorescence showed that the expression of TLR4 and p-smad2/3 proteins in H8 cells was up-regulated by the biofilm of Gardnerella vaginalis;? Adding 0.1ng/ml TGF-?1(EMT negative control concentration)to H8 cell EMT model induced by Gardnerella vaginalis biofilm,can significantly enhance the EMT of H8 cells(P<0.01).It is suggested that Gardnerella vaginalis biofilm induces EMT in H8 cells through TGF-?1/Smad pathway;? Adding TLR4 inhibitor to the H8 cell EMT model of "Gardnerella vaginalis biofilm+ 0.1ng/ml TGF-?1" can reverse the occurrence of EMT in Hs cells(P<0.01),confirming that the EMT induced by Gardnerella vaginalis biofilm in Hs cells is regulated by TLR4;? After adding TLR4 inhibitor to the Hs cell EMT model of "Gardnerella vaginal biofilm+0.1ng/ml TGF-?1",p-Smad2/3 protein was significantly down-regulated,confirming that TGF-?1/Smad is the downstream signal path,which is regulated by TLR4;[Conclusion]Gardnerella vaginalis biofilm induces EMT in Hs cells through the TLR4-TGF-?1/Smad signaling pathway.It is the main mechanism for Gardnerella vaginalis biofilm to cooperate with HR-HPV to infect human cervical epithelial cells and induce canceration.As a potential target for the prevention and treatment of cervical cancer.