| Most of the efficacy of conventional chemotherapeutic drugs is due to their ability to induce apoptosis, but the molecular mechanisms still remain unclear. Moreover, chemotherapeutic agents have obvious side effects that restricts its doses of clinical applications. So it is important to enhance the researches on mechanism of apoptosis, which contributes to analyze potential anticancer drug targets by inducing leukemic cell apoptosis, and then proceed to the development of a new specific therapeutic strategy. Resistance of leukemic cells to chemotherapeutic agents is an important factor that limits the successful treatment of leukemia. Although some multi-drug resistant related molecules have been found, such as the membrane transport-associated proteins, metabolism-related enzymes and modulating molecules of apoptosis etc, it is still difficult to explain all the mechanisms. Some reversing agents for multi-drug resistance, such as verapamil and cyclosporin A, have been used in combination with chemotherapy of leukemia. However, the effective dose of verapamil is somewhat larger than that is well - tolerant in vivo and is a major limiting factor for clinical use due to its pronounced cardiovascular effects. The cyclosporin A seems to be a more effective multi-drug resistance reversing agent than verapamil but the side-effects of cyclosporin A, such as dominant immunosuppression and toxicity to liver and kidney, restrict its clinical applications. So, further researches on mechanisms of apoptosis and multi-drug resistance becomes an interesting topic with new methods. Leukemia is influenced by many elements such as heredity and environment, so it needs to be studied with the high-throughput methods. Proteomics technology supplies a new opportunity. Proteome means the whole proteins expressed by genome in a cell, tissue or genome. Proteomics studies provide a new tool in this area. In the present study, we used some of the most popular and reliable proteomicsmethods, including the immobilized PH gradient (IPG) two-dimensional gel electrophoresis (2-DE), the peptide mass finger printing map and bioinformatics analysis to study the related proteins of apoptosis-initiation and the differentially expressed proteins between the multi-drug resistant cells HL-60/A and drug sensitive cells HL-60/S of acute myeloblastic leukemia (AML), aiming at establishing a experiment model of leukemia proteomics, revealing the mechanisms of apoptosis and multi-drug resistance and finding new potential chemotherapeutic agents and reversing agents of multi-drug resistant in AML.The cord blood transplant has been demonstrated to have good prospects in treating leukemia. Under the normal condition, the immune cells from the cord blood such as lymphocytes and NK cells can kill tumor cells, but expression of their antigens is weak in the cord blood and the cytotoxicity to tumor is low, so its anti-tumor effect decreases. As a result, an important topic for cord blood transplant is how to activate immune cells of cord blood. Oliogodeoxynucletides (ODNS) containing bacterial and viral DNA CpG (CpG-ODN) are known as an excellent immune adjuvant and can activate various immune cells subsets to play its antitumor role. But the immunological competence of lymphocytes from cord blood treated by CpG- oliogodeoxynucletides (CpG-ODN) and its effects on the leukemic cells have not been reported. The peptidoglycan and lipoteichoic acid of cell wall have directly anti-tumor function, but how CpG motif of bacterial DNA and CpG-ODN exert their direct function is unclear. The study is very significant to comprehend biology function of CpG motif comprehensively.After establishment techniques of proteomic analysis about leukemic cells, we studied the model of apoptosis initiation induced by Homoharringtonine in HL-60 cells and its some signal transduction pathways. With comparative proteomic techniques, we studied the related proteins of apoptosis initiation induced by Homoharringtonine, the differentially expressed proteins between acute myelocyte leukemia multi-drug resistant cells HL-60/A and drug sensitive cells HL-60/S, and the effect that lymphocytes from cord blood inhibits leukemic cells and induces leukemia HL-60 cells differentiation and apoptosis treated by CpG-ODN. The main contents of the study include:1. The establishment of the techniques in the proteome research of HL-60 cellsTo establish the techniques in the proteome research of leukemia cells, proteins of HL-60 cells were extracted with several kinds of solutions which have differential solubility, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the proteins extracted were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis. Protein spot detection and matching were performed using the 2-DE image analysis software. With assisted laser desorption ionization time of flight mass spectrometry ( MALDI-TOF-MS) and database searching, protein spot A from HL-60 cells was identified. The result indicated that the 2-DE maps of HL-60 cells were established, and protein spot A from HL-60 cells was identified successfully. Consequently, the establishment of the techniques in the proteome research of leukemia cells set the foundation of the researches of leukemia proteome.2 . Study on signal transduction of apoptosis initiation induced by Homoharringtonine in HL-60To Study on signal transduction of apoptosis initiation induced by Homoharringtonine(HHT) in HL-60, after establishment the model of apoptosis initiation induced by Homoharringtonine in HL-60 cells , caspase-3> Bcl-2, Bax> fas/fasL were mensurated with flow cytometry and transmission electron microscope at the apoptosis initiation phase. ERK2 and P38 expression of HL-60 cells were detected using immunohistochemistry. The result indicated the model of apoptosis initiation induced by Homoharringtonine was established in HL-60 cells, at the apoptosis initiation phase, upregulation of caspase-3, decrease of Bcl-2/Bax were observated., ERK2 expression decreased and P38 expression increased, and expression of fas/fasL had not significantly changes. Consequently, Caspase-3 , Bcl-2 , Bax and mitogenactivated protein kinases pathways were inverved in signal transduction of apoptosis initiation induced by Homoharringtonine in HL-60.3 . Comparative proteomics research of apoptosis initiation induced by homoharringtonine in HL-60 cellsTo study the related proteins of apoptosis initiation induced by homoharringtonine(HHT) in HL-60 cells, after establishment of an apoptosisinitiation model induced by HHT in HL-60 cells , proteins of untreated and HHT-treated HL-60 cells were extracted* and the two-dimensional polyacrylamide gel electrophoresis maps of the extracted proteins were established by using the immobilized pH gradient two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Proteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride Channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by Homoharringtonine. Consequently, the present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.4. Proteomics analysis between acute myelocyte leukemia multi-drug resistant cells HL-60/A and drug sensitive cells HL-60/STo study differentially expressed proteins of acute myelocyte leuemia multi-drug resistant cell line HL-60/A and drug sensitive cells HL-60/S, the two-dimensional gel electrophoresis maps of the proteins, extracted from two acute myelocyte leuemic cell lines, the multi-drug resistant cell line HL-60/A and the drug sensitive cell line HL-60/S, were established respectively. The extracted proteins were digested by enzymes and identified with the Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting was matched to available databases of proteomics on the Internet. Proteomics analysis showed 13 differentially expressed proteins in the HL-60/A cells were identified. They involve the protein disulfide isomerase precursor(PDI), the proteasomes a 1 and other proteins which are related to drug resistance or cell metabolism. So, studies on the differentially expressed proteins in the multi-drug resistant cells would be helpful to the understanding of proteomics of the drug-resistant cells and the mechanisms of multi-drug resistance.5 . The effect of lymphocytes from cord blood treated by CpG-oIigodeoxynucletides on K562 cellsTo observe the antileukemic effect of lymphocytes from cord blood treated by...
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