The Effect And Mechanism Study On ALA Photodynamic Disgnosis And Photodynamic Therapy The Malignant Brain Tumor

The people health and lives are seriously threatened by the brain tumors and the gliloma are the most frequent malignant brain tumor. The glioma cells grow infiltrativly into the normal brain tissues, especially the brain important domain or around the region. The tumors are impossible completely resected by the surgical operations and the tumors recurred easily after the operation. The radiotherapy and chemotherapy both could kill the tumor cells, but the treatment could damage the normal brain tissues meanwhile the patients had serious all over the body adverse reaction. Recently the researchers put the fluorescence spectroscopy technique into checking the tumor by autofluorescence or drug fluorescence. The minimal changes which are unable or difficultly to find by naked eyes may be develop an image. So the detection rates of precancerous lesion or minimum cancer rise significantly.Photodynamic diagnosis (PDD) involves the use of a photosensitizer in areas of high tumour concentration. The photosensitizer is activated by a particular wavelength of light and produce fluorescence. This can be used to guide diagnosis and to locate the border of tumours. The modality Photodynamic Therapy (PDT) is a local form of treatment that involves the administration of a photosensitizing agent followed by photoactivation. PDT is a local treatment involving the administration of a tumor-localizing photosensitizing drug, in this case aminolevulinic acid (ALA).The researches suggest ALA-PDD or ALA-PDT could help the doctor diagnosis and therapy brain tumor effects significantly. But how about the effect of ALA-PDD combinate with ALA-PDT? There is no one answer. It is a new method to the clinic treatment the brain tumor if we approach the combination role. On the other hand, the mechanisms of ALA-PDT brain tumor mainly concentrate into how about the tumor cell apoptosis and necrosis. With the development of autophagy the people think highly of the relation between autophagy with the tumor. Some regulating factors are shared by autophagy and apoptosis. Coordinating reasonably the autophagy/ apoptosis and the autophagy of the tumor cells are actived may help the PDT research and the development of the correlation subjects.To study the effect and mechanism of ALA- PDD and/or PDT on brain tumor, we first made the implanted rabbit VX₂ brain tumor models and ALA photodynamic guiding microsurgery and/or photodynamic therapy (PDT) on brain tumor. The tumour resection was confirmed with histological analysis. All animals were examined with MRI for presence of any residual tumour tissue. The survival time of each rabbit was recorded. We investigated the efficacy of ALA in photosensitizer VX₂ brain tumour. The C6 glioma cells were cultured and passage in vitro. The effect of ALA density and culture time on ALA-PDT C6 glioma cells. The apoptosis rate of the glioma cells were measured by TUNEL. The autophagy phenomenon were observed by MDC staining and Transmission Electron Microscope. The role of the autophagy and apoptotic mechanisms about ALA-PDT induced the death of C6 glioma cells were researched. The survival rates of the C6 glioma cells were measured by MTT when the C6 glioma cells were pretreated by the autophagy inhibitor 3-MA or the apoptosis inhibitor z-VAD-fmk. The lysomal enzyme cathepsin B proteinum and caspase-3 were measured by western-bolt to analyze the autophagy and apoptotic mechanisms on ALA-PDT induced death of C6 glioma cells. The main results were as follows:1. All treatment groups (conventional white light microsurgery group, photodynamic therapy group, PDD-guided microsurgery group and a group in which PDD-guided microsurgery was followed by PDT) showed a significantly extended survival time compared with the control group (P<0.01). PDD-guided microsurgery combined with PDT significantly prolonged survival time, compared with PDD-guided microsurgery alone (P<0.05). MRI results confirmed removal of most of the tumours by PDD-guided microsurgery combined with PDT or PDD-guided microsurgery, compared with conventional white light microsurgery group. Histological examination showed that the tumour cells were enriched in the high-intensity fluorescence region. In the low-intensity fluorescence region, there were some tumour cells with a diffuse distribution; the caryocinesia and cell density decreased significantly, even approaching the normal tissues, showing oedema and a rare abnormal vascular endothelial hyperplasia. There were no tumor cells around the nearby area. There were no fluorescence in necrotic tumor area. Autopsy result suggested tumor recurred in the operation cavity. Five cases of recurrence were observed in the conventional white light microsurgery group (50% recurrence), five in the photodynamically guided microsurgery group (25% recurrence) and three in the combined guided microsurgery and therapy group (15% recurrence).2. The survival rate of C6 glioma cells decreased as the concentraion of ALA and the time of incubation with ALA increased. The survival rates were under 50% as the concentraion of ALA was 400 ug/ml and the time of incubation with ALA was 6h. TUNEL detection showed: Some rather typical apoptotic cells were observed in 200μg/ml and 400μg/ml groups with a proportionate of 49% in the former and 62% in the later.3. MDC staining results suggested there were obvious fluorescence particle(autophagic vacuoles)behind ALA-PDT 3h. Follow the treatment time continued, the intensity increased gradually, being weak behind ALA-PDT 24h. Transmission Electron Microscope (TEM) was used to examine autophagy induced by ALA-PDT in C6 glioma cells. There were a number of dependent double membrane structures behind ALA-PDT 3h. Typical apotosis shape and autophagy lysosomal emerged behind ALA-PDT 24h. There was no necrosis shape emerging in whole ALA-PDT progress.4. The apoptosis inhibitor z-VAD-fmk attenuated the growth inhibiting effect of ALA-PDT on the C6 glioma cells (P<0.05); The survival rate of C6 glioma cells raised significantly in autophagy inhibitor 3-MA pretreatment group. The death rate of C6 glioma cells degraded significantly in the combination z-VAD-fmk with 3-MA group. (P<0.05)5. After ALA-PDT 1h, intracellular calcium fluorescence intensity was very weak by confocal laser scanning microscopy. Follow the treatment time continued, the intensity increased gradually, reaching the peak behind ALA-PDT 6h and decreasing behind ALA-PDT 12h. There were always weak flurecence in control group.The results were check by flow cytometry. Intracellular Ca²⁺ in the process of autophagy of C6 cells was significantly higher than that in the control group, especially the 20min and 6h behind ALA-PDT. In group treated with ALA-PDT plus 3MA, mean Intracellular Ca²⁺ was significantly lower than that treated with ALA-PDT alone. The difference is significantly. LC3 fluorescence distribution were found behind ALA-PDT 1h and lasted behind ALA-PDT 24h.6. Western-bolt results suggested that the cathepsin B expression and the caspase-3 activation of the C6 glioma cells increased significantly behind ALA-PDT 1h, reaching the peak behind ALA-PDT. In group treated with ALA-PDT plus 3MA, cathepsin B was significantly lower than that treated with ALA-PDT alone.Our results suggest that both microsurgeryoperation and simple PDT significantly prolonged the survival time of the VX2 tumor-bearing rabbits. Photodynamically guided microsurgery combining with photodynamic therapy reinforce the treatment effects. Their combination has the potential to be used as a rapid and highly effective treatment of metastatic brain tumours. ALA-PDT is lethal effective to C6 glioma cells, and this effect was correlated with the concentration of ALA, incubation time of C6 glioma cells with ALA .The autophagy and apotosis were under the development changes. The autophagy protect role in earlier period delay the apoptosis. 3-MA may reduce ALA-PDT induced autophagy by inhibiting increase of Intracellular Ca²⁺ in C6 cells. The increase of Intracellular Ca²⁺ in ALA-PDT-treated C6 cells may be related to autophagy induced by ALA-PDT. The results will provide the further evidence for the clinical application of ALA-PDT glioma.