| SECTION 1:Morphological studies on apoptosis of bladder tumor cell line T24induced by LAK and bacillus Calmette-Guerin-activated killer cellsObjectives: Bacillus Calmette-Guerin-activated (BCG) is a very potent antitumor Biological Response Modifiers (BRM) in the intravesical therapy of superficial bladder cancer and has become clinically established. But the mechanisms of action is still not clear. In the investigation, we study the T24 morphological characters of apoptosis, which is induced by the two key effector cells: lymphokin-activated killer cells (LAK) anil BCG-activated killer cells (BAK). Anddeveloped a method that acridine orange/ethidium bromide(AO/EB) double fluorescent dye assay are applied directly in 96-w ell microtiter plates under a fluorescence microscope for analyzing apoptosis of adherent cell line and photographing.Methods: Peripheral blood mononuclear cells (PBMCs) from heparinized blood of healthy human donors were obtained by Ficoll-paque centrifugation. Cells were adjusted to a concentration of 2xl06/ml in RPMI-1640 medium containing 10% fetal calf serum. Reconstituted lyophilizate of BCG (3.75x104colony-forming units/ml) was added, and the cells were cultured for 7 days in six-well microtiter plates at 37"C and 5% CO: to generate BAK cells. LAK cells were generated in parallel with human native IL-2 (800 units/ml). Unstimulated cultured PBMCs served as controls (NK group).The bladder tumor cell line T24 was cultured in 96-well microtiter plates at a concentration of 5*103/well for 24h, after which the effector cells were added at effector-to-target cell ratio of 25:1, and coincubated for 2h, 4h, 8h and 12h as indicated in the figure legends. All assays were performed in triplicate,then removed the effector cells. 20j.il AO/EB double fluorescent dyes (lOOmg/L) were added to wells. Two minutes later, Plates were placed upside down under fluorescence microscope with emission wavelength of 360-440nm for visualizing and analysis, then photograph using Kodak 400 film with Leica MPS 60 camera. Further investigation was performed that period of time of effector-to-target incubation is 4h and cell imaging was observed immediately, after 4h or after 12h. From the photograph, we evaluate apoptosis in terms of apoptotic index (AI) and the amount of adherent cells per visual field (a visual field equals to a photograph of 200times). Cell morphology are also determined by H&E-stained adherent cell monolayer, scanning electron microscope (SEM) and transmission electron microscope(TEM). All the data were expressed in the form of graph and figure with the aid of SPSS 10.0 software.Results: BAK, LAK and NK can kill target T24 cell line through the apoptosis manner of death. Viewed by fluorescence microscopy, viable cells appear to have a bright green nucleus with intact structure while apoptotic cells exhibit a bright green nucleus showing condensation of chromatin as dense green areas. Late apoptotic cells and necrotic cells will stain with both AO and EB, however EB produces the highest intensity emission. Late apoptotic cell cells have an orange nucleus showing condensation of chromatin and necrotic cells display an orange nucleus with intact structure.1. In the investigation of groups different period of effector-to-target coincubation time, we showed that LAK is the most strong antitumor immune cell. AI is 21.13%, 40.12%, 35.09%, 46.43% and the amount of adherent cells per visual field is 66, 57, 36, 22 in 2h, 4h, 8h, 12h groups, respectively. BAK shows antitumor activity later, AI is 7.73%, 10.67%, 14.6%, 10.68% and the amount of adherent cells is 176, 137, 131, 126 in 2h, 4h, 8h, 12h groups, respectively. Here NK shows weak antitumor activity, AI is 11.33%, 8.47%, 7.27%, 10.59% and the amount of adherent cells is 155, 152, 149,150 in 2h, 4h, 8h, 12h groups, respectively. In control group without PBMCs, the mean AI is 3.49% and the amount of adherent cells is 189.2. We next investigated the 4h period of time of effector-to-target incubation...
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