纳米化Photosan光动力疗法治疗胆囊癌的实验研究
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摘要
第一部分纳米化Photosan的制备与表征
目的制备负载光敏剂Photosan的空心氧化硅纳米粒子(以下简称纳米化Photosan),并初步探讨纳米化Photosan的表征。
方法采用一步湿化学方法制备负载Photosan的空心氧化硅纳米粒子。
结果成功制备了负载Photosan的空心氧化硅纳米粒子。
结论采用一步湿化学方法成功制备了负载Photosan的空心氧化硅纳米粒子,实现了光敏剂Photosan的纳米化。
第二部分普通Photosan及纳米化Photosan对体外培养人胆囊癌细胞GBC-SD的光动力灭活作用
目的观察普通Photosan和纳米化Photosan对体外培养人胆囊癌细胞GBC-SD的光动力灭活作用。对比普通Photosan和纳米化Photosan的治疗效果及最佳治疗参数。
方法1.倒置显微镜下随时观察GBC-SD细胞治疗前后的形态变化以及生长状况。2.MTT比色分析法测定普通Photosan及纳米化Photosan对胆囊癌细胞系GBC-SD的杀伤作用。3.Annexin V-FITC/PI双标记法检测细胞凋亡与坏死。
结果1.普通Photosan-光动力疗法(PDT)治疗后4小时即出现GBC-SD细胞生长状况欠佳；纳米化Photosan-PDT治疗后2小时出现GBC-SD细胞生长状况欠佳。治疗后48小时细胞几乎全部死亡。
2.MTT比色分析法测定显示：1)单纯使用光敏剂或单纯使用光照均不能对GBC-SD细胞的生长产生抑制作用；2)相同参数设置下纳米化Photosan较普通Photosan对GBC-SD细胞的生长抑制作用更强；3)各自最佳参数设置下纳米化Photosan较普通Photosan对GBC-SD细胞的生长抑制作用更强；4)纳米化Photosan比普通Photosan对GBC-SD细胞的生长抑制作用起效更快。
3. Annexin V-FITC/PI双标记法检测显示,普通Photosan-PDT和纳米化Photosan-PDT对GBC-SD细胞的杀伤作用均以细胞凋亡为主；在各自最佳参数设置下,纳米化Photosan-PDT比普通Photosan-PDT引起的GBC-SD细胞总凋亡坏死率高。
结论1.普通Photosan及纳米化Photosan均需要配合光照才能发挥光动力疗法的治疗效果。
2.纳米化Photosan-PDT比普通Photosan-PDT对人胆囊癌细胞系GBC-SD有更强、更快的杀伤作用。
3.普通Photosan-PDT和纳米化Photosan-PDT对GBC-SD细胞的杀伤作用均以细胞凋亡为主。
第三部分Photosan及纳米化photosan对胆囊癌移植瘤的光动力疗效
目的建立裸鼠人胆囊癌GBC-SD荷瘤模型,研究普通Photosan及纳米化Photosan光动力疗法对肿瘤生长的抑制作用。
方法1.建立裸鼠人胆囊癌GBC-SD细胞移植瘤模型。2.观察普通Photosan及纳米化Photosan对裸鼠人胆囊癌移植瘤的光动力治疗效果及对裸鼠的不良反应。
结果1.人胆囊癌GBC-SD细胞接种成功率100%,10-12天肿瘤体积达0.1-0.15cm。
2.从PDT治疗后2天起治疗组(普通Photosan-PDT治疗组及纳米化Photosan-PDT治疗组)与对照组肿瘤体积就存在着统计学差异,而普通Photosan组与纳米化Photosan组比较从PDT治疗后第6天开始出现统计学差异。治疗组裸鼠移植瘤的重量显著小于对照组,且纳米化Photosan组显著小于普通Photosan组,均有统计学意义。
3.普通Photosan-PDT及纳米化Photosan-PDT治疗仅使裸鼠发生短暂表皮细小糠屑样物。
结论1.普通Photosan及纳米化Photosan均对人胆囊癌荷瘤裸鼠模型中肿瘤的生长有明显的抑制作用,后者对胆囊癌的杀伤效果明显优于前者,起效时间明显早于前者。
2.普通Photosan及纳米化Photosan给药后裸鼠无严重短期不良反应发生。
第四部分纳米化Photosan光动力治疗胆囊癌凋亡途径的探讨
目的探讨纳米化Photosan光动力治疗胆囊癌的凋亡途径。
方法1.实时荧光定量PCR检测survivin、Bcl-2、半胱氨酸天冬氨酸蛋白激酶-8、-9(caspase-8、caspase-9)基因mRNA水平的改变。2. Western-blot免疫印迹法检测survivin、Bcl-2、caspase-8、caspase-9蛋白的表达差异。
结果实时荧光定量PCR检测及Western-blot检测均显示：纳米化Photosan-PDT治疗后,survivin在治疗早期表达明显下调,治疗晚期表达明显上调；Bcl-2在治疗早期和治疗晚期表达均明显下调；caspase-8治疗早期和治疗晚期的表达与对照组无显著差异；caspase-9在治疗早期表达明显上调,治疗晚期表达明显下调。
结论1. caspase-9依赖的线粒体途径可能是纳米化Photosan光动力治疗诱导GBC-SD细胞凋亡的主要途径。
2. caspase-8介导的外源性凋亡途径可能不是纳米化Photosan光动力治疗引起GBC-SD细胞凋亡的主要途径。
3. survivin可能在纳米化Photosan光动力治疗时参与caspase-9依赖的线粒体途径诱导GBC-SD细胞凋亡。
4.Bcl-2在纳米化Photosan光动力治疗GBC-SD细胞过程中可能参与复杂的凋亡途径。
Part One The Preparation and the Mechanism of Photosensitizer-loaded Hollow Silica Nanoparticles
Objective 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 vitro
Objective 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 GBC
Objective 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-PDT
Objective 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.
Conclusion
1. 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.
目的制备负载光敏剂Photosan的空心氧化硅纳米粒子(以下简称纳米化Photosan),并初步探讨纳米化Photosan的表征。
方法采用一步湿化学方法制备负载Photosan的空心氧化硅纳米粒子。
结果成功制备了负载Photosan的空心氧化硅纳米粒子。
结论采用一步湿化学方法成功制备了负载Photosan的空心氧化硅纳米粒子,实现了光敏剂Photosan的纳米化。
第二部分普通Photosan及纳米化Photosan对体外培养人胆囊癌细胞GBC-SD的光动力灭活作用
目的观察普通Photosan和纳米化Photosan对体外培养人胆囊癌细胞GBC-SD的光动力灭活作用。对比普通Photosan和纳米化Photosan的治疗效果及最佳治疗参数。
方法1.倒置显微镜下随时观察GBC-SD细胞治疗前后的形态变化以及生长状况。2.MTT比色分析法测定普通Photosan及纳米化Photosan对胆囊癌细胞系GBC-SD的杀伤作用。3.Annexin V-FITC/PI双标记法检测细胞凋亡与坏死。
结果1.普通Photosan-光动力疗法(PDT)治疗后4小时即出现GBC-SD细胞生长状况欠佳；纳米化Photosan-PDT治疗后2小时出现GBC-SD细胞生长状况欠佳。治疗后48小时细胞几乎全部死亡。
2.MTT比色分析法测定显示：1)单纯使用光敏剂或单纯使用光照均不能对GBC-SD细胞的生长产生抑制作用；2)相同参数设置下纳米化Photosan较普通Photosan对GBC-SD细胞的生长抑制作用更强；3)各自最佳参数设置下纳米化Photosan较普通Photosan对GBC-SD细胞的生长抑制作用更强；4)纳米化Photosan比普通Photosan对GBC-SD细胞的生长抑制作用起效更快。
3. Annexin V-FITC/PI双标记法检测显示,普通Photosan-PDT和纳米化Photosan-PDT对GBC-SD细胞的杀伤作用均以细胞凋亡为主；在各自最佳参数设置下,纳米化Photosan-PDT比普通Photosan-PDT引起的GBC-SD细胞总凋亡坏死率高。
结论1.普通Photosan及纳米化Photosan均需要配合光照才能发挥光动力疗法的治疗效果。
2.纳米化Photosan-PDT比普通Photosan-PDT对人胆囊癌细胞系GBC-SD有更强、更快的杀伤作用。
3.普通Photosan-PDT和纳米化Photosan-PDT对GBC-SD细胞的杀伤作用均以细胞凋亡为主。
第三部分Photosan及纳米化photosan对胆囊癌移植瘤的光动力疗效
目的建立裸鼠人胆囊癌GBC-SD荷瘤模型,研究普通Photosan及纳米化Photosan光动力疗法对肿瘤生长的抑制作用。
方法1.建立裸鼠人胆囊癌GBC-SD细胞移植瘤模型。2.观察普通Photosan及纳米化Photosan对裸鼠人胆囊癌移植瘤的光动力治疗效果及对裸鼠的不良反应。
结果1.人胆囊癌GBC-SD细胞接种成功率100%,10-12天肿瘤体积达0.1-0.15cm。
2.从PDT治疗后2天起治疗组(普通Photosan-PDT治疗组及纳米化Photosan-PDT治疗组)与对照组肿瘤体积就存在着统计学差异,而普通Photosan组与纳米化Photosan组比较从PDT治疗后第6天开始出现统计学差异。治疗组裸鼠移植瘤的重量显著小于对照组,且纳米化Photosan组显著小于普通Photosan组,均有统计学意义。
3.普通Photosan-PDT及纳米化Photosan-PDT治疗仅使裸鼠发生短暂表皮细小糠屑样物。
结论1.普通Photosan及纳米化Photosan均对人胆囊癌荷瘤裸鼠模型中肿瘤的生长有明显的抑制作用,后者对胆囊癌的杀伤效果明显优于前者,起效时间明显早于前者。
2.普通Photosan及纳米化Photosan给药后裸鼠无严重短期不良反应发生。
第四部分纳米化Photosan光动力治疗胆囊癌凋亡途径的探讨
目的探讨纳米化Photosan光动力治疗胆囊癌的凋亡途径。
方法1.实时荧光定量PCR检测survivin、Bcl-2、半胱氨酸天冬氨酸蛋白激酶-8、-9(caspase-8、caspase-9)基因mRNA水平的改变。2. Western-blot免疫印迹法检测survivin、Bcl-2、caspase-8、caspase-9蛋白的表达差异。
结果实时荧光定量PCR检测及Western-blot检测均显示：纳米化Photosan-PDT治疗后,survivin在治疗早期表达明显下调,治疗晚期表达明显上调；Bcl-2在治疗早期和治疗晚期表达均明显下调；caspase-8治疗早期和治疗晚期的表达与对照组无显著差异；caspase-9在治疗早期表达明显上调,治疗晚期表达明显下调。
结论1. caspase-9依赖的线粒体途径可能是纳米化Photosan光动力治疗诱导GBC-SD细胞凋亡的主要途径。
2. caspase-8介导的外源性凋亡途径可能不是纳米化Photosan光动力治疗引起GBC-SD细胞凋亡的主要途径。
3. survivin可能在纳米化Photosan光动力治疗时参与caspase-9依赖的线粒体途径诱导GBC-SD细胞凋亡。
4.Bcl-2在纳米化Photosan光动力治疗GBC-SD细胞过程中可能参与复杂的凋亡途径。
Part One The Preparation and the Mechanism of Photosensitizer-loaded Hollow Silica Nanoparticles
Objective 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 vitro
Objective 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 GBC
Objective 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-PDT
Objective 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.
Conclusion
1. 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.
引文
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