Photodynamic Therapy Treatment Of Human Glioma Mediated By 5-aminolevulinic Acid

Glioma derives from neural epithelium, which gets the annual incidence rate of(7 ~ 10) / 10 million people, is the most common malignant tumor of intracranial tumors, accounting for 40% to 50%. Most of these patients are with poor prognosis, such as patients with glioblastoma, their average survival time is only no more than 1 year, only a few of these patients can get long term survival. Most of the clinical treatments of glioma are integrated programs, including surgery, radiotherapy and chemotherapy, but the overall effect is not satisfied,because the survival rate in glioma-patients has no significant increase over the past decade, the 5-year-survival-rate of astrocytoma-patients is still fluctuating about 30%.Photodynamic Therapy (PDT) is a new treatment which appeared in the early 80s of the 20th century and gradually developed in last decade, with administering some specific chemical substances,the target tissue can concentrate these substances selectively.Then, with the activation of certain wavelengths of light,these specific chemical substances produce photodynamic damage or destruction to the target tissues. This feature makes Photodynamic therapy very suitable for adjuvant therapy of tumors.it has been reported in foreign literatures that PDT had been successfully used in the threatment of skin cancer and gastrointestinal cancer.It is a bran-new adjuvant therapy in the field of cancer treatment, alongside with surgery, chemotherapy, radiation therapy.The subject objective: To research PDT treatment of human gliomas mediated by 5-aminolevulinic acid(5-ALA), a new generation of photosensitizer, and to research the possible mechanism of the treatment for the purpose of using PDT treatment of cerebral glioma. Methods: In vitro experiment,collecting U251 cells of logarithmic growth phase, then launch PDT with 5-ALA,after the PDT,then to calculate the viability of U251 cells in a CCK-8 method, and, explore the possible mechanism of U251 cell death via apoptosis staining with Hoechst 33258 and Annexin-V- PI staining flow cytometry. Results: 1.The human glioma cells(U251 cells) grew well via conventional cell culture method of recovery and cultivation.2.By fluorescence microscope: the U251 glioma cells cultured with 5-ALA together produced photosensitizer protoporphyrin (PpIX). And protoporphyrin (PpIX) mostly pitch in the cytoplasm and the cell membrane, a little of which pitch in the nuclear membrane, and there had no PpIX production inside the cell nucleus.3.5-ALA itself had no cytotoxicity to the U251 cells.4. Not given any 5-ALA,only given the irradiation of laser light,the U251 cells had no significant difference in survival rate. 5.In the same conditions ,the cell survival rates were significantly different with different incubation-time of 5-ALA before PDT.Incubated for 4 to 6 hours before PDT, making cell survival minimum.6.When 5-ALA was in a certain range of low concentration ,5-ALA PDT-mediated cytotoxicity is direct proportion to its concentration, but when 5-ALA was in the concentration of 2.5mmol/l,there came the saturation.7. The same conditions, the laser energy got more powerful ,the effect of 5-ALA-mediated-PDT was more obvious, but when the total energy got the scale of 45 J/cm2,there came the saturation. 8.Hoechst 33258 staining found apoptosis in PDT groups,and there was no phenomenon of apoptosis in U251 cells from non-PDT groups.9. Using Annexin V-FITC-PI double staining flow cytometric analysis showed that: both of the apoptosis rate and the apoptosis + necrosis rate were significantly increased in PDT groups than that of the negative control groups.At the same time, there was massive necrosis cells in PDT groups.In summary, our results demonstrated that 5-ALA-mediated PDT can be effective in killing human glioma cells,with a lot of inflammatory death tumor cells,there also was apoptosis phenomenon.We believe that with the developing research on the mechanism of how can the PDT induce apoptosis,it will be greatly promoted the improvement of PDT and benefit to the society.