A Preliminary Study On The Relationship Between Several Infection Factors And Oral Lichen Planus

Objectives Oral lichen planus(OLP)is a common chronic inflammatory disease of the oral mucosa,and its exact etiology and pathogenesis are not yet clear.Long-term OLP can undergo malignant transformation and eventually become oral cancer.Erosive OLP is more prone to cancer Recently,studies have suggested that microbial infections play a role in pathogenesis of OLP.The purpose of this study is to investigate the HPV infection status,oral fungal microbial change and the detection status of four clinical infection indexes(treponema pallidum,HIV,hepatitis B,hepatitis C)in the blood and to analyze the relationship between these factors and OLP.The expected results may help to provide new ideas for understanding the causes and pathogenesis of OLP.Methods:(1)Collection of research objects:①Collection of tissue samples:Patients enrolled weer those who visited the Department of Stomatology,Yan’An Hospital,affiliated to Kunming Medical University for the first time from September 2017 to December 2019.All OLP patients met clinical manifestations and pathological features.They were divided into erosive type(group A,9 cases)and non-erosive type(group B,15 cases)according to clinical manifestations.During the same period of time,20 cases of healthy nonkeratinized mucosal tissues around the impacted tooth as a control group(R group,20 cases)were also collected.These tissues in the R group were from patients visiting the oral surgery outpatient department to remove the impacted teeth.②Collection of saliva samples:Collected 5ml whole saliva of research objects that spontaneously flowed out after fasting(including non-consumption of food and water)for 2 hours before biopsy specimen extraction.The samples were immediately removed and stored in liquid nitrogen for late use;③Collection of blood samples:Collected 5ml of peripheral venous anticoagulant blood from patients in fasting state with clinical diagnosis of OLP who visited Department of Stomatology,Yan’An Hospital affiliated to Kunming Medical University during the same period of time.Samples were sent to clinical lOLPoratory for testing.(2)Research methods:①Envision immunohistochemical technique was used to detect the expression of p16INK4a protein in groups A,B,and R.Nest PCR technique was used to detect the existence of HPV DNA;②High-throughput fungal sequencing of saliva samples of groups A,B and R was conducted to analyze the changes of oral fungal microorganisms in OLP patients;③The four clinical infection indexes(Treponema pallidum,HIV,hepatitis B and C)in the blood of OLP patients were tested according to the clinical inspection process by the clinical lOLPoratory.(3)Statistical analysis:①P16INK4a was lassified in accordance with its positive staining ratio,and the positive rate was calculated;nest PCR test was conducted and verified by sequencing.Use SPSS19.0 statistical software to perform chi-square test on the final result;②Used Qiime software for Alpha diversity and Beta diversity statistics;used SPSS 19.0 software to count the OTU sequencing number,Alpha diversity index and Beta diversity index.When comparing two sets of continuous variOLPles that follow a normal distribution,an independent sample T test was used;one-way analysis of variance was used when comparing three or more groups.For PCA R software was used for drawing,and for the remaining group difference.Graph-Prism 6 software was used for drawing;③The positive rate of four pathogen infection indexes in patients’ blood was counted.All statistical results are statistically significant with P<0.05 as the difference.Results:(1)Results of Envision immunohistochemistry technology for p16INK4a detection:①Only one case of erosive OLP lesions was observed with obvious brownish yellow staining in the nucleus and cytoplasm.That is,p16INK4a positive expression.The remaining tissues were negative expression.After statistical analysis,there was no statistically significant difference in protein expression between group OLP and group R,group A and group B(P>0.05).(2)Nest PCR technology for HPV detection and sequencing results:Only 1 case of erosive OLP tissue showed target band after gel electrophoresis.The band undergone gel recovery sequencing,and it was confirmed to be HPV 16 subtype after gene bank comparison.After statistical analysis,there was no statistically significant difference in protein expression between group OLP and group R,group A and group B(P>0.05).(3)Sequencing results of saliva fungi in OLP patients:Compared with group R,Shannon index decreased and Simpson index increased in oral fungal microorganism in OLP group.PCA shows that the sample distances of groups OLP and R are relatively close.Fungal OLPundance varies at all levels.The OLPundance of ascomycota and pullulancs tausonia increased significantly.At the family and genus levels,the OLPundance of each fungus was significantly reduced.There is no statistical difference between Shannon index and Simpson index between group A and group B.There were major fungal microorganisms with different OLPundances at the phyla,family,genus and species levels,but they showed no significant difference.(4)Results of blood infection indexes test:TP and HIV were not detected.One OLP sample had HBV infection,and the detection rate was 2.13%;one sample was positive for HCV OLP,and the detection rate was 2.13%.Conclusion:(1)After detection of tissue p16INK4a protein expression by Envision immunohistochemistry and the presence of HPV DNA in samples by nest PCR,we found no correlation between OLP and HPV/p16INK4a.(2)Through high-throughput sequencing,it was found that the diversity of fungal flora in saliva samples of OLP patients is lower than that of healthy people.However,OLP patients’ fungal group spectrum composition is similar with that of that of normal population,and the number of Candida is less than that of the normal population.There is no difference in the composition and number of major fungal microorganisms between erosive and non-erosive OLP.(3)By analyzing the four infection indexes of OLP patients’ blood,we found that TP and HIV were not detected and the detection rate of HCV and HBV infection was also low in OLP patients.