The Establishment And Application Of EBV Quantitative Detection Method In Thymoma

Objective:At present,the relationship between EBV(Epstein-Barr virus,EBV)infection and the onset of thymoma and thymus-associated autoimmune diseases has become a research hotspot.Previous studies have found that there are some differences in the research results on the relationship between them,and the interaction mechanism of action is not yet clear.Free EBV DNA in the blood of patients with EBV-related diseases.This experiment aims to quantitatively detect the free EBV DNA in the blood samples from patients with thymoma and thymus-related diseases,and explores the relationship between the number of EBV DNA copies in blood and thymoma and thymus-related diseases.In addition,the relationship between EBV DNA copies number and the classification and stage of thymoma was explored to provide theoretical support for further guidance in treatment,judgment of efficacy and prognosis.At the same time,plasmids containing EBV DNA are constructed to provide support for subsequent molecular mechanism research.Methods:Quantitative Real-time PCR(q PCR)technology was used to establish a quantitative detection method of EBV DNA to detect the number of copies of EBV DNA in the blood of patients with thymoma and thymus-related diseases in this experiment.In addition,the samples are grouped according to different standards to explore the differences between the EBV DNA copies in the blood of patients with thymoma and myasthenia gravis and the control,and then explore the correlation between EBV infection and the onset of thymoma and myasthenia gravis.In addition,this experiment uses flow cytometry to detect the relationship between EBV DNA copies and lymphocyte subtypes in patients with thymoma and thymus related diseases.At the same time,a plasmid deriving from the EBV DNA amplified in the blood of patients with EBV infection and a plasmid containing the standard sequence of EBV DNA are constructed to the further study of the mechanism of EBV DNA in EBV-related diseases.Results:36 samples are detected by q PCR,including 23 samples from thymoma patients and 15 samples from patients with myasthenia gravis.The results suggest that EBV DNA copies in the blood of thymoma samples are different from that of the control,suggesting that EBV is actived in thymoma.The EBV DNA copies of patients with myasthenia gravis was compared with the control,and concluded that EBV infection involvs in the occurrence of myasthenia gravis still need study.However,the analysis of thymoma WHO pathological classification and Masaoka-Koga staging subgroups of EBV DNA copy number difference is not obvious.In addition,the results of flow cytometry showe that with the increase of EBV DNA copies,the expression of CD19 and CD20 molecules on the surface of B cells increased.No significant correlation is found between EBV DNA copies and the ratio of Th17 or Treg cells,but the Th17 / Treg ratio in the patient ’s blood increases with the increases of the copies in the patient ’s blood sample.The construction of plasmids containing the standard EBV Bam HI-W gene and the Bam HI-W gene derived from patients infected with EBV thymoma in this experiment,laying a foundation for the subsequent study of the mechanism of EBV infection in the pathogenesis of thymoma and myasthenia gravis.Conclusions:QPCR method for the quantitative detection of EBV DNA copies is successfully established in thisexperiment.Furthermore,clinical thymoma samples were tested with good repeatability and accuracy.This method lays foundation for the quantitative detection of EBV DNA copies during the treatment of patients with thymoma and thymus-associated autoimmune diseases.It is beneficial to the implementation of clinical antiviral therapy and provides a reference for the prognosis of antiviral therapy for patients with thymoma and myasthenia gravis.After EBV infection,the expression of lymphocyte surface membrane molecules in patients has a certain trend with the change of the active state of EBV,which provides help for the subsequent research on the mechanism of EBV infection and thymoma and thymus-related autoimmune diseases.