| Background! Hepatocellular carcinoma (HCC) is one of the most commonmalignancies in the world, especially in sub-Saharan Africa and Southeast Asia. It has been the second cause of cancer death in China. About 140,000 people die of this death in China. Though regular ultrasound examination can detect early small HCC and there are many therapeutic modalities for HCC, the therapeutic results remain unsatisfactory. HCC is localized and can be successfully treated with surgery. Disseminated metastases and highly invasive tumors, hower, are frequently not curable by surgery and /or radiation therapy, leaving chemotherapy as the principal method of treatment. A major problem with chemotherapy is its lack of tumor specificity and inablity to localize to tumor cell deposits. As a result, chemotherapy, the major treatment for metastatic cancers, cannot be used at high enough does to completely destroy metastatic tumor because at these doses healthy tissue is also destroyed. The same problem applies to raditon therapy.The ideal cancer therapy should have the potency to eradicate systemictumor at multiple sites in the body, as well as the specificity to discriminate between neoplastic and non-neoplastic cells. In both of these respects, immunotherapy is potentially an attractive approach. In addition, the immune response exhibits acute immunological memory, making it capable of responding to its targets over a long period of time. Vaccination against HCC, therefore, has the potential to be a highly efficious and specific treatment for metastatic cancer and may even provide long-term protection against recurrence of disease or latent outgrowth of pre-existing.T cells play a critical role in antitumor immunity. T cells recognize tumor antigens via peptides associated with the MHC class I and class II. The complexes of peptides and MHC class I molecules are expressed on the surface of virtually all nucleated cells, suggesting that most (if not all) aberrant tumor proteins are available as targets of cytolytic T lymphocytes (CTLs). Several methods have been used to identify CTL epitopes of tumor antigens. If the amino acid sequence of a protein antigen is known, overlapping peptides 8-10 amino acids in length can be synthesized and screened as CTL targets; this approach has led to the discovery of a number of CTL epitopes of tumor proteins. CTL epitopes may also be identified subsequent to the search for MHC-binding motifs and measurement of the affinity of potential antigenic peptides for MHC molecules. If the protein source is unknown, isolation of peptides from MHC class I molecules followed by purification and sequencing provides a direct way of identifying CTL epitopes. Genetic approaches, such as the screening of DNA expression libraries, provide another attractive approach and have led to the discovery of the majority of human tumor antigens recognized by CTLs.Mass spectrometry has in the last decade been accepted as a keyanalytical technique in protein chemistry. The two ionization techniques presently widely used in protein studies are matrix-assisted laser desorption/ionization (MALDI) electrospray ionization (ESI) . By virtue of the ability of mass spectrometry based techniques to analyze complex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopes important in both the humoral and cellular immune responses.The aim of this research is to characterize HHC associated peptides in order to understand antitumor immunity and development novel tumor vaccines based on these peptide epitopes.Methods: Firstly, we used IFN- y to induce the expression of HLA-I on HHCC cells , expression of HLA-Imolecules was assessed by flow cytometry using the mouse mABs. Then,HLA-associated peptides were extracted by mild acid wash of viable cells, next,peptides were isolated by Sephadex G-25 and fractionated by RP-HPLC on an Applied Biosystems seperation system. Fourthly, peptides were reconstituted of T cell epitopes with T2 cells, then,Cytotxicity...
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