Identification Exosomes Of Hepatoma Cell And Proteomics Detection

Nowadays, AFP, a kind of liver cancer serum marker, is used the most widely on clinic. However, the effect of using AFP to detect liver cancer is limited, so there is needed to explore a new valuable and useful marker. In 2013, James E. Rothman, Randy W. Schekman, and Thomas C discovered the mechanism of cellular regulatory for vesicular trafficking. They won a Nobel Prize in Physiology or Medicine because the uncovering of the mystery of the mechanism of intracellular transport. As an important class of vesicles in human body, exosomes has become a hot topic on researching biomarkers. Because of the active substances such as specific proteins, lipids, RNA, microRNA that exosomes contain, exosomes play an important role in cellular communication, cellular migration, and some other aspects. Currently, multiple biomarkers are found within exosomes; for example, detecting EGFR, MET, ALK and BRAF within exosomes can be used as the markers of lung cancer, which provides a new idea of studying liver cancer biomarkers.Protein is the direct manifestation of life functions, proteomics investigate the characteristics of protein on the expressed level, post-translational modification, the interaction between protein and protein, and some other broad-scaled studies. Thereby, obtaining information about the disease happening and cellular metabolism on the level of protein. On the process of exploring exosomes, the research of exosomes proteomics has rarely been reporting. Therefore, this study was to investigate a new program for isolating the exosomes from hepatoma cells. Modifying ultra-centrifugal separation to purity the exosomes from hepatoma cell SMMC-7721, normal liver cell HL-7702, HCC serum, and control serum; also, identifying them based on morphology and specific proteins. LC-MS/MS mass spectrometry was used to analyzed hepatocellular carcinoma cell and the main protein within its exosomes. Furthermore, analyzing the related functions and providing the foundation for further study on exosomes within hepatoma cell.CHAPTER ONE ISOLATION AND IDENTIFICATION EXOSOMES OF HEPATOMA CELLObjective:Investigating the methods of extracting and identifying exosomes from hepatocellular carcinoma cell and serum; and preparing for the research on the markers of exosomes.Method:Continuous Continually adapting serum-freed hepatoma SMMC-7721 cells and collecting cellular supernatants. Using ExoQuick and ExoQuick, ultrafiltration, and modified ultracentrifugation to isolate and purify the exosomes from supernatant of hepatoma cells. ExoQuick and ultracentrifugation were used to extract serum exosomes; TEM was used to observe the form of exosomes; Nanosight was used to analyze the size of particle; Western blot was used to detect the expression of specific protein Alix, CD63, and CD9 within exosomes; SDS-PAGE electrophoresis was used to analyze the protein profiling of hepatocellular carcinoma cell and serum exosomes.Results:The exosomes that were extracted by ExoQuick and ExoQuick and combining with ultrafiltration had low concentrations and poor purity. Modified ultracentrifugation was able to separate exosomes that were high purified. The shape of exosomes from hepatoma cells were round or oval under TEM. Nanosight analyzed that 72.16% particle within exosomes had peak diameter of 122 nm and the diameter of 30-150 nm. Western blot confirmed that the protein expression of Alix, CD63, and CD9 could be found in both supernatant and extracted exosomes. SDS-PAGE electrophores is clearly showed that high-abundance macromolecular structured proteins were less in exosomes of hepatoma cells than that in hepatoma cells and serum.Conclusions:Using continuously adapted serum-freed cells to culture and combine with ultracentrifugation could extract high purified exosomes. High-abundance macromolecular structured protein within exosomes that come from hepatoma cells had less interference, which were helpful for the further study on the markers of exosomes.CHAPTER TWO PROTEOMICS HEPATOMA CELL EXOSOMESObjective:Applying LC-MS/MS mass spectrometry proteomic strategy to analyze and identify hepatoma cells SMMC-7721 and its major protein within exosomes. Moreover, using bioinformatics to analyze their features.Method:Isolating and purifying the exosomes from hepatoma cells SMMC-7721. Utilizing LC-MS/MS mass spectrometry to identify the major protein within hepatoma cells SMMC-7721 and its exosomes, and selecting the common expressed protein. DAVID system was used on the common expressed protein for classifying them into GO classification and analyzing them into signal transduction pathway; String system was used to analyze the interaction of network protein.Results:After retrieving human uniprot database, and using high-confidence (greater than 95%) peptide,4922 hepatoma cells SMMC-7721 proteins,1568 SMMC-7721 exosomes proteins, and 1035 common proteins between them were detected. Among them, peptide spectra hits, the relatively high protein which was PSMs> 40, were 70.4 of them were membrane protein, which were myosin 9 (MYH9), retinoic acid-induced protein 3 (GPRC5A), the integration of β-1 (ITGB1), humanized recombinant protein (GALNT2); The analysis of the classification for these 70 proteins showed that the majority of proteins belong to organelles or membrane proteins (31%), combined class (60%), and transferred/carried proteins (28%). Based on the description of signaling pathway for these 70 protein,2 were the important signaling pathways that related to hepatic carcinoma, which were Spliceosome and Pathways in cancer. String analyzed the interaction between 70 proteins. The displayed node proteins were DHX9, PRKDC, HSP90AA1, ITGB1, AGRN, RUVBL1, EEF2.Conclusions:Hepatoma cells SMMC-7721 and its exosomes had common expressed proteins. The membrane protein that exosomes contained, the 2 important signaling pathways, and the critical node proteins within the interaction between proteins were the emphasis on further researches.