| Osteosarcoma is the most common malignant tumor of bone with high degree of malignant and poor prognosis. The 5-year survival rate increased to 70% after the introduction of new adjuvant chemotherapy in patients with osteosarcoma, but the survival rate has been in a plateau with no significant improvement over the past 20 years. Therefore, it is an urgent clinical need for a new adjuvant treatment of osteosarcoma which is more safe, effective, and able to overcome the disadvantages such as chemotherapy-resistant.Photodynamic therapy (PDT) is a novel treatment modality. With the local or systemic injection of a particular photosensitizer which is selectively gathered in the lesion, the treatment site then be irradiated using the appropriate wavelength of visible light. The biological killing effect is achieved by the combination of light, photosensitizer, and oxygen molecules in the treatment site.5-aminolevulinic acid (5-ALA) is one of the main issue in the field of PDT. 5-ALA has been widely studied and clinically applied in the skin cancer, esophagus, gastrointestinal tract cancer and lung cancer. The role of 5-ALA-PDT in osteosarcoma has not been systematic studied. For this study the possibility of using 5-ALA-PDT in the treatment of the osteosarcoma was evaluated in vitro and in vivo, and the anti-tumor mechanism was preliminary studied.Chapter one In vitro study on the effect of 5-ALA-PDT on osteosarcoma cell linesObjectiveTo investigate the intracellar metabolic procedure of 5-ALA to Ppâ…¨, the cytotoxicity of 5-ALA alone, the cytotoxicity of laser irrigation alone, the cytotoxicity of 5-ALA-PDT and it's optimal interaction parameters.Methods1 Fluorescence microscope was applied to detect the location of Ppâ…¨after 5-ALA being intaked into MG-63 and LM8 cells.2 Fluorospectrophotometry was applied to detect the metabolic regulation of PpIX after 5-ALA being intaked into MG-63 and LM8 cells.3 MTT assay was applied to determine the the cytotoxicity of 5-ALA alone, the cytotoxicity of laser irrigation alone, the effects of different 5-ALA dose, the effects of different laser dose, and the the effects of different 5-ALA incubation time. 4 Phase contrast microscope, cells seeded HE staining and transmission electromicroscopy is applied to observe the morphological changes of MG-63 and LM8 cells after 5-ALA-PDT.Results1 Emission of red light was detected in MG-63 and LM8 cells by fluorescence microscope after exposuring to 409 nm light and PpIX merely existed intracellarly.2 Fluorospectrophotometry detected that the amount of PpIX production increased along with the increasing concentration of the 5-ALA, the 5-ALA incubation time and the concentration of the cells.The increasing slowdown when the concentration of the 5-ALA up to 2 mM and the incubation time up to 8 h.3 The results of MTT assay suggested that the 5-ALA alone(0.25mM-8mM) or the laser irrigation alone(5 J/cm2-80 J/cm2) had no significant affection on MG-63 and LM8 cells within 12 h(p>0.05).5-ALA-PDT showed a significant cytotoxicity compared to the control group(p<0.05) in MG-63 and LM8 cells, with the effects increased along with the increasing of the concentration of 5-ALA, the laser dose and the 5-ALA incubation time.The cytotoxicity effect had no significant change when the concentration of the 5-ALA up to 4 mM, the laser dose up to 60 J/cm2 and the incubation time up to 10 h(p>0.05).4 Under the phase contrast microscope, most MG-63 and LM8 cells turns apoptosis, collapse, necrosis, and thus floated off walls, the cell structure was unclear and losing their shape. Cells seeded HE staining showed the apoptosis of the vast majority of cell, the edge was unclear, the structure of nucleus no longer exists. Shrunken cell body were observed under transmission electron microscope, with the swelling of mitochondria, endoplasmic reticulum dilation, swelling, and a large number of vacuolated cytoplasm changes. Apoptotic bodies can be seen, that was particle, massive, non-dense structure of condensed chromatin distributed in the inner nuclear membrane.Nucleus pyknosis was also detected.Conclusions1 MG-63 and LM8 cells produced PpIX efficiently after intaking 5-ALA,which was mainly accumulated in the cytoplasm and nucleus.2 The 5-ALA alone or the laser irrigation alone showed no significant affection on the growth of MG-63 and LM8 cells.5-ALA-PDT showed a significant cytotoxicity on MG-63 and LM8 cells, with the effects increased along with the increasing of the concentration of 5-ALA, the laser dose and the 5-ALA incubation time. ObjectiveTo investigate the metabolic procedure of Ppâ…¨in tumor and normal tissue, the anti-tumor and anti-metastasis effect of 5-ALA-PDT through establishment model of osteosarcoma in C3H mouse.Methods1 Osteosarcoma model of C3H mouse was established through subcutaneously injection of cultured LM8 cells.2 Fluorospectrophotometry was applied to detect the metabolic procedure of Ppâ…¨in tumor and normal tissue after tail vein injection of 5-ALA.3 C3H mouses were divided randomly into six groups when the tumor diameter reached 0.6 cm-0.8 cm:the control group, the group of 5-ALA alone, the group of laser irrigation alone, the group of 5-ALA-PDT low dose(50 mg/kg), the group of 5-ALA-PDT medium dose(100 mg/kg), the group of 5-ALA-PDT high dose(150 mg/kg). Each group were treated respectively.4 Tumor volume was measured and counted on 1,3,7,10,14 days after treatment. After the last measurement, all mouses were killed and the tumor tissues were weighed.5 Tumor tissues and lung tissues of all groups were embedded in paraffin wax for pathological evaluation. Lung metastasis was also evaluated.Results1 Osteosarcoma model of C3H mouse was successfully established through subcutaneously injection of cultured LM8 cells with tumor formation rate of 97.5%. The maximum diameter of tumor nodules reached 0.6 cm-0.8 cm about 14 days after inoculation.2 Fluorospectrophotometry detected that the Ppâ…¨mainly accumulated in tumor and liver tissue, and little in other normal tissues. In normal tissues, the yield of Ppâ…¨reached it's peek 4-6 h after 5-ALA injection and then declined. In tumor tissue, the amount of Ppâ…¨reached it's peek 8 h after 5-ALA injection.3 The tumor volumes of experimental group had significant reduction compared to the control group(p<0.05). The tumor volumes between the control group, the group of 5-ALA alone and the group of laser irrigation alone had no significant difference(p >0.05).There was significant difference of tumor volume between the group of 5-ALA-PDT low dose and the group of 5-ALA-PDT medium dose or high dose(p< 0.05). The tumor weight showed no significant difference between the conrol group, the group of 5-ALA alone and the group of laser irrigation alone(p>0.05), and significant difference between the group of 5-ALA-PDT low dose and the group of 5-ALA-PDT medium dose or high dose(p<0.05).4 Pathological evaluation showed the collapse of the vast majority of cell, the edge was unclear, the structure of nucleus no longer exists, even without hematoxylin coloration, leaving only the structure of incomplete cytoplasmic in the group of 5-ALA-PDT medium dose or high dose. Lung metastasis was detected in the vast majority of mouse in the control group, the group of 5-ALA alone and the group of laser irrigation alone, and most mouse in the group of 5-ALA-PDT medium dose or high dose showed no lung metastasis. The difference was significant(p<0.05).Conclusions1 Osteosarcoma model of C3H mouse was successfully established.2 The Ppâ…¨mainly accumulated in tumor and liver tissue. In tumor tissue, the amount of Ppâ…¨reached it's peek 8 h after 5-ALA injection.3 5-ALA alone or laser irrigation alone showed no anti-tumor effect of mouse osteosarcoma.5-ALA-PDT showed significant anti-tumor and anti-metastasis effect on mouse osteosarcoma, and the 5-ALA medium dose or high dose were better than low dose.Chapter Three Preliminary study on the mechanism of anti-tumor effect of 5-ALA-PDT on osteosarcomaObjectiveTo investigate mechanism of anti-tumor effect of 5-ALA-PDT on osteosarcomaMethods1 MG-63 and LM8 cells with 5-ALA-PDT were analyzed by flow cytometry with Annexin-V-FITC/PI apoptosis detection.2 EnVision immunohistochemistry technique was applied to detect the expression of PCNA,Ki-67,VEGF,Bax and Bcl-2 protein in the control group, the group of 5-ALA alone, the group of laser irrigation alone, the group of 5-ALA-PDT low dose, the group of 5-ALA-PDT medium dose and the group of 5-ALA-PDT high dose respectively.Results1 MG-63 and LM8 cells were mainly apoptosis after 5-ALA-PDT. Flow cytometry with Annexin-V-FITC/PI apoptosis detection showed the apoptosis and necrosis rates in 5-ALA-PDT group were both significant higher than control group(p<0.05).2 Results of immunohistochemistry showed the positive expression rate of PCNA,Ki-67,VEGF and Bcl-2 protein in the control group, the group of 5-ALA alone, the group of laser irrigation alone and the group of 5-ALA-PDT low dose was(++), and in the group of 5-ALA-PDT medium dose or high dose was(+). The positive expression rate of Bax protein in the control group, the group of 5-ALA alone, the group of laser irrigation alone and the group of 5-ALA-PDT low dose was(+), and in the group of 5-ALA-PDT medium dose or high dose was(+-++).Conclusions1 5-ALA-PDT can induce apoptosis of osteosarcoma cells as well as kill them directly, and mainly in the former way.2 The anti-tumor effect of 5-ALA-PDT on osteosarcoma was probably achieved by inhibiting the proliferation of osteosarcoma cells, inhibiting angiogenesis and inducing apoptosis, but still need for further validation by expanding the number of samples, increase getting the tumor tissue at different time, and using molecular biological methods.
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