| Leukemia, a malignant tumor derived from hemotological system, belongs to a malignant clone disease of hemopoietic stem cell. Studies of immunology have indicated a close relationship between occurrence and development of leukemia and immunity of organism. Immunotherapy can completely clear up residual leukemic cells and cure the disease.Immunoreaction is firstly an antigen capture by antigen presenting cells (APC), then APC elaborates and handles the antigen and transfers the processed antigen to T/B lymphocyte to induce a series of specific immune responses of organism. So the effect of APC is the first element in immune response. Dendritic cell is a professional antigen presenting cell which is the most functional and can uniquely activate naive T lymph cell. So DC is an immunoreactive initiator of body and it occupies a central place in inducting and regulating immune response. Most of studies have indicated only when mature in function can DC produce a marked antigen presenting effect. A great quantity of experiments have proved the combination of cytokines rhGM-CSF and rhIL-4 can induce leukemic cell into a DC, and the leukemia-derived DC is generally defective in function, so in DC mediated anti-leukemia effect, it is of great significance to find highly effective and harmless drugs to promote mature of leukemia-derived DC.Leukemic multidrug resistance is a major reason for refractoriness and recrudescence. If we can induce multidrug resistant leukemic cell into a DC which is named with multidrug resistant leukemia-derived DC and promote its maturity with effective and harmless drugs, multidrug resistant leukemia-derived DC not only carries the special antigens of leukemia but also can present the special antigens toimmune system to kill corresponding leukemic cells. At the same time, it can reverse indirectly leukemic multidrug resistance. In clinic, better therapeutic effects to chronic myelocytic leukemia have been achieved with the application of rhIFN a -2b and HHT. Most studies have indicated rhIFN a -2b and HHT can promote the maturity of DC derived from chronic myelocytic leukemia (CML), but there are no reports that rhIFN a -2b, HHT or the combination of rhIFN a -2b and HHT can promote the maturity of DC derived from multidrug resistant leukemic cell K562/A02.Various studies have displayed that CpG oligonucleotide can activate normal blood- or bone marrow-derived DC through up-regulating the expression of MHC-I> II molecules and costimulatory molecules CD80 and CD86, stimulating APC to secrete cytokines, and so on. A novel family of immunomodulatory oligonucleotides characterized by a phosphodiester backbone, a lenghth of six bases such as 5' -GGG A GG-3' and 5' -GGG T GG-3' named for A-ODN and T-ODN, which derived from the DNA sequence of Mycobacterium phlei(M. phlei), can stimulate T/B lymphocytes to generate cytokines. However, there are no reports whether CpGODN^ A-ODN and T-ODN can induce the maturity of multidrug resistant leukemia-derived DC.The reverse of multidrug resistance is a key point in the therapy of leukemia, and studies have shown that progestogen antagonist mifepristone (RU486) can effectively reverse multidrug resistance of such tumor cells as carcinoma, thymoma, breast carcinoma. However, there are no literature reports whether RU486 can reverse multidrug resistance of leukemic cells.In our search, we would induce multidrug resistant cell K562/A02, which derives from CML cell K562 and is resistant to ADM, to turn them into immature leukemia-derived DC with the combined utilization of cytokines rhGM-CSF and rhIL-4, and observed immunoregulation effect of these drugs or oligonucleotides such as Hffl\ rhIFNa -2b,. CpG20(^ A-ODN and T-ODN to multidrug resistant leukemia-derived DC through morphology n immunophenotype and metergasis. At the same time, we have discussed reversal mechanism of RU486 reversing multidrug resistance of K562/A02. The major research procedures and results are following: 1. Induction of dendritic cell derived from multidrug resistant leukemic cellTo study whether CML multidrug resistant cell K562/A02 can differentiate intoDC when induced by the combination of cytokines rhGM-CSF and rhIL-4, and evaluate the degree of the maturity of multidrug resistant leukemia-derived DC, we observed the morphological change of multidrug resistant leukemia-derived DC with help of the inversed microscope and the Gimsa-Wright staining, detected the change of immunophenotype by FACS and understood the functional change with allo-MLR, CTL lethal effect by MTT test and the secretion of cytokines IL-12 and IL-6 by ELISA before and after the application of cytokines rhGM-CSF and rhIL-4. Through these targets we understood the degree of the maturity of multidrug resistant leukemia-derived DC. we have found that K562/A02 can be induced into an functional immature DC, which was called K562/A02-DC-7. To further study whether rhTNF a can promote the maturity of K562/A02-DC-7, we divided it into two groups, one group added TNF a and the other added LPS, which was thought as a positive control, to the culture system for 3 days. Afterwards, we observed the maturity-promoted effect on K562/A02-DC-7 with the methods mentioned forward. We found that the number of dendritic cells, the expression of the special antigens and immunoloregulation molecules of DC increased obviously, and leukemia-derived DC showed significant stimulating effect on the proliferation of antogenic T cells after adding rhTNF a . At the same time, CTL activation has increased notably and the secretion of cytokines has visibly rised. There are no notable difference compared with adding LPS group. So, rhTNF a can promote the maturity of K562/A02-DC-7. 2. Maturity-promoted effect on multidrug resistant leukemia-derived DC after the application of HHT and rhIFN a -2bTo study the maturity-promoted effect on K562/A02-DC-7 after the application of HHT> rhIFNa -2b and HHT+rhlFN a -2b, we firstly induced K562/A02 into K562/A02-DC-7 with the method introduced in chapter 1, then separately added HHT, rhIFN a -2b and HHT+rhlFN a -2b to the culture system for 3 days. Afterwards, we observed the maturity-promoted effect on K562/A02-DC-7 of the application of HHT, rhIFN a -2b and HHT+rhlFN a -2b with the same detected methods as employed in chapter 1. We have found the HHT> rhIFN a -2b and HHT+rhlFN a -2b can all promote the maturity of K562/A02-DC-7 and we have observe the most obvious effect after the application of HHT+rhlFN a -2b. So, HHT, rhIFN a -2b andHHT+rhlFN a -2b are functionally effective in promoting the maturity of multidrug resistant leukemia-derived DC, which provides a new way for immunotherapy of leukemia.3. Maturity-promoted effect on multidrug resistant leukemia-derived DC after application of the oligonucleotides of CpG2006> A-ODN and T-ODNTo study maturity-promoted effect on K562/A02-DC-7 after application of the oligonucleotides of CpG2006> A-ODN and T-ODN, we firstly induced K562/A02 into K562/A02-DC-7 with the method employed in chapter 1, then separately added CpG2006> A-ODN and T-ODN to the culture system for 3 days, Afterwards, we observed the maturity-promoted effect on K562/A02-DC-7 of the application of CpG20(^ A-ODN and T-ODN with the same detected methods as introduced in chapter 1. We have found the CpG20(^ A-ODN and T-ODN can promote the maturity of K562/A02-DC-7, which provides a new idea for immunotherapy of leukemic multidrug resistance.4. Mechanism study of progestogen antagonist mifepristone reversing multidrug resistance of leukemiaIn order to investigate whether progestogen antagonist mifepristone (RU486) can reverse multidrug resistance of K562/A02, we observed the effect of K.562/A02 cell proliferation and the change of K562/A02 cell sensitivity to ADM before and after the application of different concentration RU486. We found RU486 could reverse multidrug resistance of K562/A02 cell. In order to further study the mechanism of RU486 reversing multidrug resistance, we studied the change of apoptosis relatation proteins, such as bcl-2> Bax and Caspase-3, with immunohistochemistry and detected the alteration of the glucosylceramide synthase mRNA expression with RT-PCR before and after the application of RU486. We have found that the possible mechanisms of RU486 reversing multidrug resistance of K562/A02 are related with regulating the expression of apoptosis relatation proteins bcl-2> Bax and Caspase-3, decreasing the mRNA expression of glucosylceramide synthase. So RU486 may be a potential reversal agent of multidrug resistance for leukemia. |
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