| Objective Leukemia is one of the common malignant tumors in our country and the leading cause of cancer in children and adolescent. High-dose chemotherapy and autologous stem cell transplantation are an useful modalities for patients with leukemia. A small proportion of these patients could be healed by chemotherapy or radiochemical therapy. However, autologous stem cell transplantation is limited by a high relapse rate, which may be attributed to the presence of residual tumor cells in autografts. Photodynamic therapy (PDT) has been used to eradicate residual tumor cells and to reduce the contamination in autografts. Light exposure can selectively kills the tumor cells with minimal toxicity to progenitor cells or stem cells because of much more uptake of photosensitizer in malignant cells than normal cells. Photodynamic gene transfection (PGT), based on the use of photosensitising compounds, which localise in the membranes of endocytic vesicles and, upon activation by light, induce photodynamical reactions, leading to the permeabilisation of the vesicular membranes and cytosolic release of the vesicular content, i.e. transfecting DNA, was said to enhance the efficiency of gene transfection. Although the primary mechanism of tumors killed by PDT is still not completely understood, it is well known that PDT can lead tumor cell cycle to arrest and apoptosis in many kinds of cells, including k562 cell line of chronic myelogenous leukemia. This thesis reports the effects of HMME-PDT in combination with bcr-abl fusion gene antisense oligodeoxynucleotide (AS ODN) in vitro on the growth of k562 cell line of chronic myelogenous leukemia and its partial molecule biology mechanisms as well. Methods 1. The human chronic myeloid, leukemic K562 cells obtained from the Department of Heamatology of Southwest Hospital of Third Military Medical University (Chongqing, China) were cultured in RPMI 1640 (Hyclone, USA) media, supplemented with 10% heat-inactivated calf serum (Hali, China) and 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified atmosphere. Cells were harvested, resuspended in phosphate-buffered saline (PBS, pH = 7.2, Hyclone) at a total concentration of 1×105, and immediately used for the experiment. Hematoperphyrin monomethyl ether (HMME,10%mg·ML-1) was purchased from Zhangjiang Co.(Shanghai ,China). A solution of 20μg·ML-1 HMME was freshly made in 0.9% sodium chloride before each experiment. Cells were collected during the exponential growth phase. Cell viability was determined at >95% before each experiment.2. In vitro cells (1×105) were plated in 96-well dishes with k562 cell line treated with HMME and FAM-labelled AS ODN simultaneously and cultured for 1.5h .The cells were then washed with RPMI 1640 and irradiated with 9 J·CM-2 low power laser from semiconductor laser (Laser Instrument Co., Southwest Normal University, Chongqing, China, wave length: 650nm, power: 15mW, light exposure time: 30 mins). Power output from the fiber was measured with a power meter (Boulder Co., USA). After irradiation, the cells were incubated with fresh medium for another 6h;3. The uptake of FAM-labeled AS ODN was detected by flow cytometry (FCM);4. The intracellular localization of FAM-labeled AS ODN was probed by fluorescence microscope;5. Typan blue exclusion test and clonogenic assay were used to observe the inhibition effects dealing with morphologic changes and drawing growth curve; 6. Cell cycle arrest was measured by flow cytometer; 7. Cell apoptosis was showed by terminal deoxynucleotidyl transferase-medited dUTP nick end labeling (TUNEL) respectively; 8. Effects of HMME-PDT and AS ODN on the expression of bcr-abl mRNA was analyzed by RT-PCR; 9. Statistics: All experiments were performed at least five times. Data were expressed as mean±S.E. Statistical analysis was performed by t-test or one-way analysis of variances (ANOVA) at SPSS10.0 and statistical significance was considered at P<0.05.Results 1. The uptake of AS ODN into k562 cells was evidently increased by photodynamic gene... |
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