Isolation And Proteome Study Of Extracellular Vesicles In Saliva

Body fluids based molecular diagnostics is routinely used in clinic,which has the merits of accuracy,simple,and rapid.As an important body fluid,human saliva could reflect the health status of our body.Due to its simple sample collection,easy preparation,low cost,human saliva has been used for the noninvasive diagnostics of diseases.Extracellular vesicles(EVs)are vesicles with membrane structures secreted by cells.They could selectively encapsulate matters such as nucleic acids and proteins from the host cells and enter the extracellular microenvironment,which play important roles in cell signaling transduction.Studies have demonstrated that they are intimately related to the occurrence,development,and metastasis of cancer,and might be used in the diagnosis and therapy of cancers.Human saliva harbors plenty of EVs and has important value for research and future application.Focused on the isolation and proteome study of EVs,the main contents of this dissertation are as follows:First,according to the principle of specific binding between enzyme and substrate,we established a saliva sample processing technique based on affinity chromatography.Combining with high speed centrifugation,a new method was developed for the enrichment of EVs from saliva.Comparing with the traditional extraction method,our results demonstrated EVs with wider size could be obtained through the developed new method.The yield was increased by 18%.The number of identified EVs proteins was increased by about 70%.Our data revealed that the enrichment efficiency of extracellular vesicles in saliva could be interfered by high abundant proteins.The enrichment method established in this chapter for EVs paved the basis for this dissertation.Second,through employing the established method,we enriched two main kinds of extracellular vesicles in saliva through optimizing the centrifugation force,one is microvesicles and the other is exosomes,respectively.Multiple characterization method including TEM,NTA,and WB were utilized to confirm and compare the salivary microvesicles and exosomes.The proteome of two types of EVs in the saliva of lung cancer patients were compared,lung cancer related proteins were revealed boh in microvesicles and exosomes.Through label free quantification analysis,4 candidate proteins in microvesicles and exosomes were discovered and were further verified by western blot method.Our data collectively demonstrated that salivary EVs harbor informative proteins that might be used for the detection of lung cancer through noninvasive way.Third,after the high abundant proteins were depleted,exosomes in human serum and saliva were enriched by commercial kit,which was easy and fast.The proteome in both exosomes was analyzed.Our results demonstrated that the number of serum exosomal proteins was about three times that of salivary exosomal proteins,while 50% of salivary EVs proteins were shared with serum.The similarity of exsomal proteins in saliva and serum of lung cancer group was higher than that of normal group and lung cancer related proteins presented in the exosomes of both body fluids.In summary,through developing method for the separation and enrichment of EVs,the proteome in salivary EVs was systematically analyzed.In total,1162 salivary EVs proteins were identified from human saliva.Systemic disease,namely lung cancer,related proteins were discovered.The feasibility of salivary EVs as biomarker source for the detection of lung cancer was explored.Based on noninvasive advantages of salivary diagnostics,salivary EVs are expected to be used for specific screening and molecular diagnosis of cancer in the future.