Preliminary Research Of Circulating Tumor Markers

Tumor markers are a classs of substances produced by tumor cells or abnormally produced by the body under the stimulation of tumors.They play an important role in diagnosis and monitoring of tumors.There are many types of tumor markers,including proteins,nucleic acids,metabolites,etc.The nucleic acid tumor markers mainly include genomic DNA mutation,mRNA expression level and peripheral blood free circulating nucleic acid.Tumor markers can be detected in tissue cells or in body fluids such as serum and plasma.Such markers that can be detected in body fluids are often referred to as circulating tumor markers.Generally,the acquisition of its specimens has the advantages of non-invasiveness and the convenience of dynamic monitoring of diseases,which has become an important content of clinical detection.Although there are a large number of circulating tumor markers that have been clinically applied,there are still some limitations in the early diagnosis of tumors.Recently,researchers have found some tumor cells in the blood of tumor patients.These circulating tumor cells that falled from the tumor's primary or metastatic sites can reflect the tumor information,and have important significance in the detection of tumors.However,circulating tumor cells are less abundant in the circulation and have high heterogeneity,which is a difficult point in their clinical application.With the development of molecular biology,researchers have also discovered circulating tumor DNA,exosomes and other substances in body fluids,They carry important biological information and play an important role in tumor diagnosis,prognosis analysis,and efficacy monitoring,which are expected to become new types of circulating tumor markers.In order to meet the needs of clinical disease diagnosis,exosomal mRNA and serum proteins were studied separately so that to seek circulating tumor markers for solid tumor and hematological malignancies.In the first part of this study,PML-RARA fusion gene which is in acute promyelocytic leukemia(APL)cell was used as an example to establish an experimental method for the detection of minimal quantity of fusion genes in APL cell-derived exosomes.By Taqman probe-based droplet digital PCR(ddPCR)technique,we detected exosomal PML-RARA fusion gene transcripts derived from acute promyelocytic leukemia(APL)cell.RNA from PML-RARA negative cell line HL60 was used as a negative control to set the limit of blank(LOB),while RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S)were used to determine the limit of detection(LOD)for long and short PML-RARA transcripts,respectively.The results show that the LOB of this ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system,respectively.The LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system,respectively.Furtherly,by ddPCR technique,we analyzed the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia(APL).We established a ddPCR-based technique for detecting fusion gene transcripts,which could be used to analyze the minimal quantity of PML-RARA transcripts derived from exosomes by absolute quantification.Our study explored a possibility of non-invasive and dynamic monitoring of the exosomal PML-RARA transcripts from APL patients serum.In the second part of the study,we explored the role of secreted protein-controlled protein(TCTP)as a tumor marker in serum.We used enzyme-linked immunosorbent assay(ELISA)to detect TCTP in serum samples from tumor patients and healthy people,in order to analyze the difference in expression of TCTP between tumor patients and healthy people.Unfortunately,due to the limitations of detection techniques,this study is currently unable to conclude the relationship between serum TCTP expression levels and tumors.