Analysis And Validation Of Differentially Expressed Genes In Refractory Periapical Periodontitis With RNA-seq Technology And Case Reports Of Selected Typical Endodontic Diseases

Background: Chronic periapical infection is a chronic inflammation of the periapical tissue that occurs due to bacterial infection.Refractory periapical periodontitis is defined as a tooth that has undergone more than one root canal treatment and still has significant clinical symptoms or destruction of periapical bone tissue,which is more complex in etiology and treatment than untreated chronic periapical infection.Transcriptome high-throughput sequencing(RNA-sequencing,RNA-seq)technology enables high-throughput detection of transcripts from all species to obtain information on the genes encoded and to accurately target them.It is a sequencing-based,high-throughput transcriptome sequencing technology that can sequence a wide range of RNAs to study their transcriptional expression.RNA-seq technology is now a powerful tool for delving into the complexity of the transcriptome and has been widely used in biological research,medical research,clinical research and drug discovery.In dentistry,RNA-seq technology has been used to study oral squamous cell carcinoma,dental caries,endodontic disease,periodontal disease,pulp regeneration and oral implants with certain achievements,but the analysis of differentially expressed genes in refractory periapical periodontitis based on RNA-seq technology is less studied.Exploring differentially expressed genes in refractory periapical periodontitis is a useful reference for the study of potential biomarkers as well as therapeutic targets in refractory periapical periodontitis.Objective: The RNA-seq technique was applied to analyse and validate differentially expressed genes in refractory periapical periodontitis,providing some theoretical basis for the study of the pathogenesis of refractory periapical periodontitis.Methods: Clinical samples were collected according to the inclusion criteria,with clinical refractory periapical periodontitis tissues in the experimental group and healthy periodontal tissues in the control group.RNA-seq was used to screen for differential genes in refractory periapical periodontitis,box plot,heat map and volcano plot were used to analyse gene expression levels,GO enrichment analysis and KEGG enrichment analysis were used to analyse gene expression functions.Real Time PCR was used to detect the expression of B cell lymphoma factor 3(Bcl3),interleukin-6(IL-6),guanylate binding protein 2(Gbp2),and V-Set domain containing T cell activation inhibitor 1(Vtcn1)in refractory periapical periodontitis,which showed the greatest difference in gene expression in response to protozoan biological function.1.Collection of clinical samplesThe experiment was approved the Stomatological Hospital Affiliated to School of Stomatology of Dalian Medical University(Approval Number : 2021006),and after obtaining informed consent from patients,healthy periodontal tissues of patients with refractory periapical periodontitis tissues removed by apical surgery and orthodontic extractions who met the inclusion criteria and each of the patients attending the Stomatological Hospital Affiliated to School of Stomatology of Dalian Medical University from Feb 2021 to Jun 2021 were selected 13 cases.2.Transcriptome chip sequencing13 refractory periapical periodontitis tissues and 13 healthy periodontal tissues each were selected clinically,of which three refractory periapical periodontitis tissues and three healthy periodontal tissues were used for transcriptome sequencing to screen for differential genes.3.Analysis of differential gene expressionBox plots and heat maps were used to analyse gene expression levels,GO enrichment analysis and KEGG enrichment analysis were used to analyse gene expression functions.4.Detection of Bcl3,IL-6,Gbp2 and Vtcn1 expression in refractory periapical periodontitisVerify the four factors with the greatest difference in gene expression in transcriptome sequencing results by Real Time PCR.The expression of Bcl3,IL-6,Gbp2 and Vtcn1 in refractory periodontitis was examined by Real-Time PCR in 10 refractory periapical periodontitis tissues and 10 healthy periodontal tissues in response to protozoan biofunction.The results of the experiments were statistically analysed using the t-test in SPSS Statistics 21.0 and were statistically different at P<0.05.Results:1.Collection of clinical samplesThirteen cases each of refractory periapical periodontitis tissue removed by apical surgery and healthy periodontal tissue from orthodontically extracted patients were selected.The age of the refractory periapical periodontitis group was 28-48 years,7males and 6 females.The control group was 21-39 years old,6 males and 7 females.There was no statistical difference in age and sex between the control and refractory periapical periodontitis groups,P>0.05.2.Transcriptome chip sequencing resultsThe results of the RNA-seq screen for differential genes showed 2540 up-regulated genes,2195 down-regulated genes and 19224 non-differential genes in refractory periapical periodontitis.3.Results of differential gene expression analysisGO enrichment analysis identified response to protozoan,cellular response to interferon-beta and response to interferon-beta as the first three major biological functions in refractory periapical periodontitis.glycosaminoglycan binding,heparin binding and receptor ligand activity as the first three major molecular functions.The first three major molecular functions are: glycosaminoglycan binding,heparin binding and receptor ligand activity.extracellular membrane-bounded organelle,extracellular matrix and symbiont-containing vacuole.containing vacuole were the first three major cellular components.The four most differentially expressed factors in the response to protozoan inflammatory pathway were Bcl3,IL-6,Gbp2 and Vtcn1.coagulation cascades and Neuroactive ligand-receptor interaction were the three major signaling pathways.4.Expression of Bcl3,IL-6,Gbp2 and Vtcn1 in refractory periapical periodontitisReal-Time PCR results showed higher expression of Bcl3,IL-6,Gbp2 and Vtcn1 in refractory periapical inflammation than in the control group,results consistent with RNA-seq results.Conclusion:Transcriptome high-throughput sequencing technology is both rapid and sensitive in screening for differentially expressed genes in refractory periapical periodontitis,supporting the screening of potential biomarkers and therapeutic targets in refractory periapical periodontitis.Objective: Through the diagnosis,treatment and prognosis of typical cases of different types of endodontic periapical disease,the students will be able to master microscopic endodontic techniques and the clinical treatment of complex variant root canals and difficult cases.Methods: Typical cases of initial root canal treatment,root canal retreatment,presence of root canal variants and central cusp fracture in young permanent teeth with root hypoplasia presented to the Dental Hospital of Dalian Medical University,School of Stomatology,were collected and the patient’s history,examination and diagnosis were recorded in detail.Microscopic pulpotomy,root canal exploration,root canal cleaning and preparation and case photographs are collected and the patient is followed up regularly to observe the prognosis.Results: Cases 1 and 2 were treated for double root canals of mandibular lateral incisors,Case 3 was retreatment of a missed lingual root canal of a mandibular cuspid,Case 4 was treated for double root canals of a mandibular first premolar,Cases 5,6 and7 were treated for a young mandibular premolar malocclusion with a fractured central cusp resulting in an underdeveloped root tip,and Cases 8,9 and 10 were treated or retreated for root canals of mandibular molars.Cases one to ten were followed up by telephone one week after root canal treatment and the patient was free of discomfort.Case 4: 6 months after endodontic treatment,the resin filling of the affected tooth was intact,with percussion pain(-)and no abnormalities on radiographs;Case 5: 6 months after pulpal blood flow reconstruction,CBCT showed a reduction in the apical shadow area of the affected tooth,an increase in periapical bone density,apical closure and thickening of the root canal wall.Case 6: 31 months after pulpal revascularization,the follow-up radiograph showed no loosening of the tooth,disappearance of a large dark shadow in the apical region,closure of the apical foramen,root growth and thickening of the root canal wall.In case 7,10 months after the pulpal blood flow reconstruction,the follow-up X-ray also showed the disappearance of dark shadows in the apical region,closure of the apical foramen,root growth and thickening of the root canal wall.The patient had no discomfort at the 4-month post-operative follow-up examination.Case 10:7 months after endodontic retreatment,CBCT showed a decrease in apical shadow area,increased bone density,percussion pain(-)and disappearance of lower lip numbness.Conclusion: Skilled use of the endodontic microscope,thorough root canal preparation and close filling of the canal,as well as knowledge of the anatomical features of the pulp and root canal and their variations,can lead to a better outcomeafter root canal treatment;the use of pulpal blood flow reconstruction in young permanent teeth with apical hypoplasia can allow the root to continue to develop and improve the crown-to-root ratio.