| Part One The Preparation and the Mechanism of Photosensitizer-loaded Hollow Silica NanoparticlesObjective To prepare the Photosan-loaded hollow silica nanoparticles and to observe their physical and chemical characterization.Methods Prepare the Photosan-loaded hollow silica nanoparticles.Results Prepare the Photosan-loaded hollow silica nanoparticles successfully.Conclusion The Photosan-loaded hollow silica nanoparticles are fit for photodynamic therapy as a kind of photosan. Part Two The deactivation of free Photosan and Photosan-loaded nanospheres-PDT in GBC in vitroObjective To observe the deactivation of free Photosan and Photosan-loaded hollow Silica nanospheres-photodynamic therapy (PDT) in gallbladder cancer cells (GBC-SD) in vitro. Compare the therapeutic effect and the optimal treatment parameters between the free Photosan-PDT and Photosan nanosphere-PDT.Methods1. The Changes of morphology and growth condition of the GBC-SD before and after PDT were observed by inverted microscope.2. The deactivation of free Photosan and Photosan-loaded hollow Silica nanospheres-PDT in GBC-SD were determined by MTT assay.3. Cell apoptosis and necrosis was detected by Annexin V-FITC/PI double marking method.Results1. The GBC-SD showed poor growth4hours after free Photosan-PDT while they showed poor growth just2hours after Photosan-loaded nanospheres-PDT. The GBC-SD almost totally died48hours after PDT.2. The MTT assay showed that, photosensitizer or light could not take effects themselves only on the growth inhibition rate of GBC-SD. Under the same parameters, the Photosan-loaded nanospheres-PDT could make larger effect on the growth inhibition rate than the free Photosan-PDT. Under the optimal parameters of their own respectively, the Photosan-loaded nanospheres-PDT could make larger effect on the growth inhibition rate than the free Photosan-PDT. Photosan-loaded nanospheres-PDT could take effect much faster than the free Photosan-PDT.3. Annexin V-FITC/PI double marking method showed that, the free Photosan-PDT and Photosan-loaded nanospheres-PDT both killed GBC-SD cells through apoptosis mainly. Under the optimal parameters of their own respectively, the total rate of apoptosis and necrosis of GBC-SD was higher in the Photosan-loaded nanospheres group than the free Photosan group.Conclusion1. Both of the free Photosan and the Photosan-loaded nanospheres required illumination to exert photodynamic therapeutic effect.2. Photosan-loaded nanospheres-PDT showed stronger and fast effects on the growth inhibition rate of GBC-SD cells.3. The free Photosan-PDT and Photosan-loaded nanospheres-PDT both induced apoptosis mainly in killing GBC-SD. Part Three The photodynamic effect of free Photosan and Photosan-loaded nanospheres in the nude mice bearing GBCObjective To establish the model of nude mice bearing GBC-SD and to investigate the tumor growth inhibition of free Photosan and Photosan-loaded hollow Silica nanospheres-photodynamic therapy.Methods1. The model of nude mice bearing GBC-SD was established.2. The photodynamic therapeutic effect and the side effects of free Photosan and Photosan-loaded nanospheres in nude mice bearing GBC-SD were investigated.Results1. All nude mice were transplanted with GBC-SD successfully, tumor lesions all reached at0.1-0.15cm3in10-12days after transplantation.2. The effects of PDT in2therapeutic groups (including free Photosan group and Photosan-loaded nanospheres group) were significantly better than the control group. It was significantly different in volumes between the therapeutic groups and the control group2d after treatment. And it was significantly different in volumes between the free Photosan group and the Photosan-loaded nanospheres group6d after treatment. The weight of transplantation tumor in therapeutic groups were significantly less than the control group, while the Photosan-loaded nanospheres group was significantly ligher than the free Photosan group.3. The nude mice just had chaff crumbs kind things in surface for a short time after PDT.Conclusion1. There were significantly inhibition effects of tumor growth on transplantation GBC-SD tumors of nude mice under Photosan or Photosan-loaded nanospheres-PDT. The effect of the latter was significantly stronger and faster than the former.2. Free Photosan and Photosan-loaded nanospheres had no apparent toxicity themselves, so the nude mice showed no severe adverse reaction after dosing. Part Four The investigation of the apoptotic pathway of GBC in photosan-loaded nanospheres-PDTObjective To investigate the apoptotic pathway of gallbladder cancer cells (GBC-SD) in Photosan-loaded hollow Silica nanospheres-photodynamic therapy (PDT).Methods1. The mRNA expression of survivin, Bcl-2caspase-8and caspase-9were detected by real-time fluorescent quantitative PCR.2. The difference of protein expression and kinase activation state of survivin, Bcl-2caspase-8and caspase-9were detected by western-blot method.Results The expression level of survivin in early stage was significantly lower than that in the control group, while it was higher in the advanced stage. The expression level of Bcl-2both in early stage and advanced stage were lower than that in the control group. The expression level of caspase-8both in early stage and advanced stage had no significantly distinction with the control group. The expression level of caspase-9in early stage was significantly higher than that in the control group, while it was lower in the advanced stage.Conclusion1. The mitochondrial pathway dependently on caspase-9mightbe the dominant apoptotic pathway of Photosan-loaded hollow Silica nanospheres-photodynamic therapy.2. Caspase-8induced extrinsic pathway might not be dominant in Photosan-loaded hollow Silica nanospheres-PDT-induced apoptosis.3. Survivin might participate in the mitochondrial pathway dependently on caspase-9in Photosan-loaded hollow Silica nanospheres-PDT.4. Bcl-2might participate complicated apoptotic pathway in Photosan-loaded hollow Silica nanospheres-PDT. |
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