【作者】 赵冰；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:研究从海葵组织中提取的海葵毒素(Phyllodiscus semonii toxin, PsTX)对人神经胶质瘤细胞(U251)的凋亡诱导作用及其可能机制。方法:MTT法检测PsTX对肿瘤细胞的增殖抑制率;原位末端脱氧核糖苷肽转移酶分析法(TUNEL)及DNA Ladder法检测PsTX对肿瘤细胞的凋亡诱导作用;RT-PCR、免疫组织化学染色分别检测Bax mRNA及Fas蛋白在U251细胞中的表达。结果:PsTX对人神经胶质瘤细胞具有明显的生长抑制及促凋亡作用,PsTX诱导Fas在人神经胶质瘤细胞膜上表达增高及诱导Bax mRNA增加。结论:PsTX可能通过Fas及Bax途径诱导人神经胶质瘤细胞凋亡。更多还原

【Abstract】 Apoptosis induction of cancer cells is regarded as the most important mechanism of anticancer agents. Previous studies have demonstrated that a wide range of anticancer agents, including chemotherapeutic agents, hormones, and various biological compounds, induced apoptosis in malignant cells in vitro. At present, there is a growing interest in deriving potential anticancer agents from the marine ecosystem. Some anticancer agents were derived from the venoms of Cnidarias. For example, Weinheimer had derived the prostanoide from one kind of Cnidaria as early as in 1969. Kikuchi et al also derived the prostanoide from another kind of Cnidaria, and named it as clavulone. Kikuchi et al reported that this clavulone demonstrated a very strong effect on the cell growth of tumors by arresting the G1-phase cells and inhibiting the S-phase cells of leukemia.Cnidarias are primitive animals such as sea anemones, corals, jellyfishes and Hydra. Sea anemones Phyllodiscus semonii (so called Unbachiisogintyaku in Japanese), is abundant in the Okinawa sea of Japan in summer season. Although the toxins isolated from several species of sea anemone are known to be cytotoxic and cytolytic, the underlying mechanism still remain unclear. Recently, sea anemone toxins were reported to include the phospholipase A2 (PLA2) and intracellular Ca2+ concentration increasing substances, both of which were found having the ability to induce the apoptosis by other researchers. Therefore, the cytotoxic and cytolytic effect of the venoms of sea anemone might be mediated by inducing the apoptosis in the malignant cells.The process of apoptosis is often mediated by Fas, which is a type I membrane protein, belonging to the receptor family of the tumor necrosis factor (TNF). Fas is also expressed in malignant glioma cells making the cellsusceptible to Fas antibody-mediated apoptosis. Induction of Fas ligand and up-regulation of Fas after administration of the cytotoxic drugs have been observed in leukemia and glioma cells. The Fas mediates a death signal in the cell.The purpose of this study was to investigate whether Phyllodiscus semonii toxin (PsTX) from nematocysts of sea anemone, could induce apoptosis in U251malignant glioma cells, and if so, to elucidate the possible mechanism involved. Cell viability was estimated by MTT assay. Apoptosis was evaluated using TdT (terminal deoxynucleoridyl transferase)-mediated dUTP nick-end labeling (TUNEL) method and DNA gel electrophoresis. PsTX had a cytotoxic effect to glioma cells. The fragmented DNA and large numbers of cells became TUNEL positiveU251cells treated with PsTX. Furthermore in order to study the mechanism of apoptosis induced by PsTX, the Fas protein expression was measured using flow cytometry. The Fas protein expression increased in the tested U251 cells at 4h after administration of PsTX in our studies. Therefore, the PsTX might cause the apoptosis through expression of Fas protein in the glioma cells. It was suggested that PsTX is promising as a potential candidate for tumor therapy.Cell viability assay by MTT method The cells were plated using 96-well plates, and each of which had a total number of 104 cells. After incubation for 24 h, each cell was respectively treated with PsTX at 2.5, 5, 10, 20, 40, and 80 ug/ml. After the cells were cocultured with each fraction for 48 h, the cell viability was assayed by MTT method as reported previously (Sun et al., 2000).Measurement of apoptotic cells Gel electrophoresis and the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labeling(TUNEL) methods were used to investigate whether PsTX induces the apoptosis or not.DNA gel electrophoresis The Gong`s modified method was used in this assay. Cellular DNA was extracted from the U251 cells treated with PsTX at the dose of 2.5, 5, 10 mg/ml for 24 h treated with PsTX at the dose of 10, 20, 40 ug/ml for 6 h.DNA Nick End Labeling by the TUNEL Method:Cells treated with PsTX for 6 h were harvested. Then, those cells were washed using the 0.2 M phosphate-buffered saline and fixed with methanol/acetate for 10 min. At last, they were stained by In situ Cell Death Detection kit, which can detect the apoptotic cell nuclei in situ labeling with a TdT-mediated incorporation of fluorescein-dUTP into the 3` ends of DNA fragments. The TUNEL positive cells were measured and photographed using a confocal laser scanning microscope.Effect of PsTX on cell growth of glioma Figure 1 showed the growth curve of U251 cells treated with PsTX at various dosages. U251 cells were treated for 48 h. The IC50 of PsTX was approximately 3.5 ug/ml for C6 cells, 24 ug/ml for ECV304 cells and 70 ug/ml for U251 cells under our experimental conditions. the cell growth of the U251 cells was inhibited 50% in treatment only of a dose of 80 ug/ml PsTX.Expression of Fas Fas expression was examined using flow cytometry in U251 cells treated with a dose 40 mg/ml PsTX. The peak FITC-fluorescence shifted to the right when the cells were treated with PsTX for 4 h.Results In present study, we demonstrated firstly that PsTX, which isolated from sea anemone Phyllodiscus semonii, has a cytotoxic effect to glioma cells .Furthermore the fragmented DNA and large numbers of cellsbecame TUNEL positive in U251 cells treated with PsTX. The Fas/Fas ligand (FasL) system is a major regulator of apoptosis. Fas is expressed on the surface of cell membranes in a variety of normal tissue cells and malignant cells including glioma cells. The Fas protein expression increased in the glioma cells at 4h after administration of PsTX in our studies. Therefore, the PsTX might cause the apoptosis through expression of Bax and Fas in the glioma cells. Indeed calcium, a major factor in many cellular signaling pathways, has long been implicated in apoptosis since many of the nucleases activated during this form of cell death appear to require calcium. Therefore, more than one kind of substance, which can induce the expression of Fas and Bax possibly existed in PsTX. Our results suggests that sea anemone venom may be a useful tool for treatment of glioma after further study.Conclusion:1、PsTX has the obvious growth inhibitory action to the glioma cells.2、PsTX induction glioma cells occurs apoptosis3、PsTX induces Bax mRNA and the Fas protein expresses on the glioma cells advances, indicated PsTX induction glioma cells occurs apoptosis possibly through the pathway of mitochondia way and the death acceptor.4、This research also prompts PsTX is promising as a potential candidate for tumor therapy.更多还原