| Objective: The pathogenesis of carcinoma is a suspensive great problem ,and at present carcinoma is cured by surpressing the proliferation rate of carcinoma . The proposition and rapid development of the study on apoptosis provide undountedly a new point of view to observe the formation and therapy stragency of neoplasm.There is a mounting evidence to indicate that the disequilibrium of apoptosis is the main reason during the formation of neoplasm and that it is a novel and effective method for curing carcinoma to induce the apoptosis of tumor cells.Many observations which came from experiments conducted in vivo or in vitro have confirmed that chemotherapeutic drugs exerted its anticancer activity by inducing apoptosis of tumor cells.It will establish the basis of theory and experiment for developing new types of anticancer drugs inducing the apoptosis of tumor cells to study deeply the moleculer mechanism of apoptosis and expression regulation of gene related to apoptosis. Based on the above-mentioned cognition, in our study human ovarian cell line HO-8910 which was established by our domesticexperiment was treated by cisplatin in vitro to elucidate the mechanism of chemotherapeutic drugs induced apoptosis and its role in cancer chemotherapy and explore the change of the expression protein of the gene p53 related to apoptosis.Our study will provide a screening pattern for new types of anticancer drugs and provide the basis of theory and experiment for gene therapy.Methods: Following exposure to cisplatin at different concentrations for different times,the HO-8910 cells were harvested and investigated by applying invert microscope to observe the situation of apoptosis, cytochemistry stain to observe the morphological chang of apoptotic cells, gel electrophoresis to assayed the DNA degradation, immunohistochemical analysis to examine the change of of P53 protein expression.Part of smears were stained with TUNEL(terminal-deoxynucleotidyl transferase UTP nick end labeling ) and the numbers of apoptotic cells counted by the two methods was compared.Results :1. morphological observation of apoptotic cells(1) When exposed to 9.9ug/ml cisplatin for 25h, under the invert microscope HO-8910 cells which were in adherent cell cultures lost contact with their neighbours,shrinked and rounded up,detached from the surface of the culture bottle and become smaller,the outlin of cells became clearer,the ability of cells to attach to the wall weakened ,cells were easyto exfoliate from the bottle wall,these were characteristic changes of apoptotic cells.(2)Under light microscope, compared to the normal adherent cells, apoptotic cells which were stained by Gimsa showed the morphological features associated with apoptosis ,that is ,they were smaller than normal cells,but had intact plasma membrane, their chromatin was condensed, apoptotic bodies could be observed in the cytoplasm.2. The situation of electrophoresis of DNA extracted from HO-8910 cellsElectrophoresis was performed on agarose gel .DNA extracted from HO-8910 cells which were exposed to cisplatin at 9.9ug/ml for 25h showed characteristic "DNA ladder" pattern,this demostrated the treatment of cisplatin activated intracellular endonuclease,the enzyme digested DNA into fragments whose molecular weights were integer multiples of a subunit:the length of subunit-about 180 base pairs.As a control,DNA from routine-cultured cells without exposition to cisplatin showed a clear strap nearby the adding sample hole.3. The counting situation of apoptotic cells At the same drug concentration,the number of apoptotic cells increased progressively with prolonged time of drug incubation.(P<0.05);at the same incubation time, the number of apoptotic cells exposed to high concentration was higher than that of low concentration.These suggested that afterexposure of the human ovarian carcinoma cell line, HO-8910,to cisplatin,there was a time- and dose-dependent increase in the apoptotic cells during the inducing course.Exposed to 9.9ug/ml for 30h, the nu...
|
PDF Full Text Request