Effects Of Helicobacter Pylori Infection On The Invasion And Proliferation Of Human Periodontal Fibroblasts

BackgroundIn 1989,Krajden et al.first discovered Helicobacter pylori in oral plaque biofilm and saliva,and then in 1991,Desai et al.also detected Helicobacter pylori in dental plaque,which confirmed that this pathogen can exist in the oral cavity,and started the research on Helicobacter pylori in the oral cavity.Subsequently,through different detection methods,scholars found that Helicobacter pylori could be colonized in suival plaque,subgingival plaque,saliva,mucosa and other parts in the oral environment,and the detection rate of subgingival plaque was relatively high Helicobacter pylori has flagellum,adhesion hormone,virulence protein,LPS and other virulence factors,which can cause immune and inflammatory damage to the host.Now a number of studies show that oral Helicobacter pylori engraftment stomach outside storage,largest in clinical research and system analysis related results found that periodontitis sites of Helicobacter pylori detection rate is higher,Helicobacter pylori infection and periodontitis has certain correlation,the existence of Helicobacter pylori in the oral cavity is one of the risk factors of chronic periodontitisPeriodontal pocket epithelium is the only barrier of bacterial biofilm and connective tissue in periodontal inflammation.The intradermal epithelium usually has a surface that erodes or ulcerates,exposing underlying inflammatory connective tissue.Periodontitis is a chronic disease in which the repair of collagen fibers plays an important role.Periodontal fibroblasts in the periodontal connective tissue are easily damaged by the original microorganisms of periodontal disease,resulting in the inhibition of proliferation capacity or morphological characteristics.Therefore,this experiment aims to explore the effect of Helicobacter pylori infection on the invasion and proliferation of human periodontal fibroblasts,and to explore the possibility of its pathological damage mechanism in periodontal tissue.Materials and Methods1.Culture and identification of Helicobacter pylori SS1 and human periodontal fibroblastsHelicobacter pylori SS1 was donated by professor Sun Yong from the department of gastroenterology,Nanfang hospital.Colombia blood plate containing 6%fresh sheep blood was prepared and H.pylori SS1was inoculated into the blood plate,placed in microaerobic environment for 2-3 days.Healthy premolars aged between 14 and 25 years were collected from the stomatology department of Nanfang hospital.The periodontal membrane was scraped for primary culture of periodontal fibroblast.When the cell culture reached 80%.subculture was used for the next experiment or cryopreservation.2.Establishment of Helicobacter pylori invasion model and its electron microscopic observationThe invasive model was established by gentamicin protection experiment.Add the corresponding amount of bacterial solution to the cell culture medium according to the MOI=100:1 of the complex number of infections.After co-culture for 2,4,6,8 and 10 h,the bacteria were incubated with gentamicin-containing(100 μg/mL)medium for 2 h to kill the Helicobacter pylori.The cells were washed,digested and collected.The cell lysate was gradient diluted on the coating plate,and micro-aerobic culture was conducted for 6 d to count the colonies.The invasion model was established by calculating the invasion efficiency,and the invasion situation and cell morphological changes were observed by electron microscopy.3.Effect of Helicobacter pylori infection on cell proliferationThe invasion models with high and low concentrations were established and incubated with gentamicin(100 g/mL)for 2 h to kill the Helicobacter pylori.After co-culture for 6 h,the cells were fixed with 4%formalin for 20 min,and the expression of Ki-67 was detected by progressive incubation of 0.2%Triton X-100 osmosis,serum blockade,Ki-67 and DAPI antibodies.CCK8 kit was used to detect the proliferation of 96-well plate cells for 7 consecutive days.4.Influence of Helicobacter pylori infection on cell cycle and its mechanismThe invasion model with high concentration and low concentration was established and incubated with gentamicin(100 g/mL)for 2 h to kill the Helicobacter pylori.In vitro invasion model after adding 70%cold ethanol fixed cells,detected by flow cytometry.Total RNA was extracted and transcribed into cDNA.The transcription of Cdc25C,CDK1 and cyclinB1 was determined by SYBR Premix Ex Taq(Takara).After protein extraction,After protein extraction,expressions of Cdc25C,cdc25c-s216,CDK1,cdk1-y15 and cyclinB1 were detectedResults1.Helicobacter pylori SS1 can invade into human periodontal fibroblasts,which has an effect on cell morphologyIn this study,primary culture and isolation of normal periodontal fibroblasts were successfully carried out,and an in vitro model of Helicobacter pylori SS1 invading cells was constructed.In the 6 h invasion model,the invasion efficiency of Helicobacter pylori was better.Part of periodontal fibroblasts were observed to become round after Helicobacter pylori infection by inverted microscope,and Helicobacter pylori adhered to the surface of the cell membrane.Transmission electron microscopy showed that Helicobacter pylori could invade into cells,and was surrounded by vacuoles and located in the cytoplasm.The number of intracellular vacuoles increased,and Helicobacter pylori in vacuoles presented bacillus and coccus.2.After invasion,Helicobacter pylori SS1 can and lead to G2 block by Cdc25C/CDKl/cyclinBl cascade reaction and inhibit cell proliferationAs can be seen from the results of CCK8 method and Ki-67 protein immunofluorescence staining,after invasion,cell proliferation was inhibited.Flow cytometry showed that the G2 phase content increased gradually with the infection concentration(P<0.05),and the G2 phase was significantly blocked.qRT-PCR and Western Blot detection of cell cycle related regulatory proteins in G2 phase showed that,the transcription and expression of Cdc25C,CDK1 and cyclinB1 proteins in human periodontal fibroblasts were disorders,leading to G2 phase block.Conclusion1.In this experiment,an in vitro model of H.pylori SS1 was constructed,which has a good invasion rate and can better simulate the effect of different concentrations of H.pylori SS1 on human periodontal fibroblasts2.The results of transmission electron microscope and inverted microscope showed that Helicobacter pylori SS1 could adhere to and invade human periodontal fibroblasts.After the invasion,Helicobacter pylori was located in the cytoplasm and surrounded by vacuoles,and the cell morphology changed from flat to round.The number of intracellular vacuoles increased,and the Helicobacter pylori in the vacuoles presented bacillus and coccus.3.After infection,the proliferation of periodontal fibroblasts was inhibited,and the higher the concentration was,the more obvious the ability of growth inhibition was,indicating that the standard strain SS1 of helicobacter pylori had a damaging effect on normal periodontal fibroblasts.4.By detecting the transcription level and protein level of cell cycle-related regulatory proteins in the G2 phase.We found that,by affecting the regulation of Cdc25C/CDK1/cyclinB1 pathway,the standard strain SS1 of helicobacter pylori leads to G2 phase block,obstructed mitosis and inhibited cell proliferation.