| Chronic myelogenous leukemia (CML) is a clonal neoplastic disorder of pluripotent hemopoietic stem cell. A cheimeric BCR/ABL, which is formed by characteristic reciprocal chromosomal translocation, has been identified. The natural course of CML can be divided into three sequential phases: a chronic phase, an accelerated phase and a terminal acute/blastic phase. No evidence exists that treatment with chemotherapy, IFNa and hemopoietic stem cell transplantation etc. has changed the natural history of this disease in the majority patients. The phenomenon of that microvascular density (MVD) in CML chronic phase and blastic phase, which is higher than normal, is significantly higher than that in CML patient with allogeneic bone marrow transplantation suggests that angiogenesis might play an important role in the pathogenesis of CML. The investigation of the expression profile of angiogenesis regulation factors, vascular endothelial growth factor (VEGF), and their pathologic role in CML progress will provide the new treatment for CML. This research includes four aspects: (1) Expression profile of VEGF and its receptors in CML bone marrow cells and K562 cells. The mRNA expression level of VEGF, VEGFR-1(Flt-1) and VEGFR-2(KDR) in CML bone marrow cells and K562 cells was measured by semiquantitative RT-PCR, the protein expression of VEGF in K562 cells was detected by immunohistochemistry, and the secretion of VEGF from K562 cells, HL60 cells and human umbilical vein endothelial cells (HUVEC) was determined by ELISA method. (2) Evaluation of anti-apoptosis effect of VEGF on the human chronic myelocytic leukemia cell line K562. The effect of VEGF on K562 cell apoptosis, which was induced by As2O3, was analyzed through morphologic study, DNA fragmentation agarose gel electrophoresis and flow cytometry DNA ploidy analyse, and the effect of VEGF on the expression of bcl-XL, Bax and caspase-3 in K562 cells was determined by Western blot method. Meanwhile, RT-PCR technique was used to demonstrate differentiation of mRNA expression of bcl-XL, Bax and ZK7, an anti-apoptosis related gene. (3) Investigation of effect of VEGF on Angiopoietin-2 mRNA and metalloproteinase (MMP) -9 expression in K562 cells. RT-PCR and Western blotting were used to detect and optimize the VEGF's effect on ang-2 mRNA expression and MMP-9 in K562 cell. (4)In vitro inhibition of K.562 cell proliferation by VEGF antisense deoxyoligonucleotide(ODN). A 19bp VEGF antisense-ODN, which encoded on human VEGF 6-24bp with sulfo-modification, was transfected into K562 cells by liposomal transfection. The effect of K562 cell growth was observed compare to sense-ODN. Alteration of VEGF mRNA and protein expression in K562 cells elicited by antisense-ODN with different concentrations was determined by RT-PCR and Western blotting, and VEGF secretion from K562 cell was detected by ELISA, respectively. The effect of antisense and sense-ODN with series dose on P210 protein expression in K562 cells and cells apoptosis, which was induced by As2O3, was analyzed by flow cytometry. The results showed: (1) Both of VEGF, Flt-1 present in malignant hemotologic disease cell line, including K562, HL-60, U937, CA46 and bone marrow cell form CML patient, while KDR is identified in K562 and CML bone narrow cell. The mRNA expression of VEGF was down-regulated after the transplantation. VEGF expression was positive in cytoplasm of K562 cells. The VEGF concentration from K562 cells was significantly higher than that from HUVEC (P<0.01). (2) A garose gel electrophoresis demonstrated DNA apoptosis fragment by As2O3 inducement. And flow cytometry cell cycle analysis revealed the dose-dependent elevation of sub-Gl apoptosis. The suppression of K562 cell apoptosis by dose-dependent VEGF stimulation, whereas cell cycle was no significant changes (P>0.05), was associated with up-regulation of mRNA and protein expression of bcl-XL, and down-regulation of protein expression of Bax in K562 cell. Therefore and the activation of pro-Caspase-3, the precursor of Caspase-3, was inhibited or redu... |
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