脊柱图像引导放射治疗对脊髓生物安全性影响的动物实验研究
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摘要
目的：
第一部分：建立犬精确脊柱图像引导放射治疗（Image guided radiation therapy，IGRT）模型，筛选脊柱IGRT在确保脊髓生物安全性的前提下，最大的放疗总剂量。第二部分：基于正交设计原理，通过MRI、肌电图检测放疗后脊髓损伤，探讨脊柱IGRT计划中的：不同水平的放疗射野、分割剂量、剂量率、射线角度对脊髓损伤的影响程度，以优化组合出脊髓生物安全性最好的放疗方案。第三部分：基于正交设计原理，通过HE染色、透射电镜、TUNEL法、免疫组织化学法及RT-PCR法检测放疗后脊髓损伤，探讨脊柱IGRT计划中的：不同水平的放疗射野、分割剂量、剂量率、射线角度对脊髓损伤的影响程度，以优化组合出脊髓生物安全性最好的放疗方案。
方法：
第一部分：选用雄性成年比格犬6只，随机数字表法随机分组，模拟犬胸7～12椎体及附件转移瘤，以IGRT方式分别对6只比格犬胸7～12椎体及附件给予50、60、70、80、90和100Gy剂量的放疗，于放疗3个月后活杀取材，取相同部位、相同位置的胸7～12节段椎体骨组织、脊髓材料。采用HE染色观察不同放疗剂量组骨组织的形态学的改变，透射电镜观察不同放疗剂量骨细胞超微结构改变。采用HE染色观察不同放疗剂量组脊髓神经细胞形态学的改变，透射电镜观察脊髓神经元细胞、脊髓神经髓鞘超微结构改变，采用TUNEL法观察不同放疗剂量组脊髓神经元细胞凋亡情况。第二部分：脊柱IGRT计划中包括：放疗射野5、7、9野3水平；分割剂量16Gy、20Gy2水平；剂量率4Gy/min、6Gy/min2水平；射线角度等角度、不等角度2水平，采用L12（3×23）正交试验表对以上因素进行分配，共需12只比格犬。重复两次实验，共需36只比格犬。选用雄性成年比格犬36只，随机数字表法随机后，在5周内对其各犬胸7～12椎体行总剂量为80Gy的IGRT。并于放疗前、放疗开始后每2周进行常规MRI及弥散加权成像（diffusion-weighted imaging，DWI）检测；于放疗前、放疗后12小时、1天、1周、2周分别检测皮质体感诱发电位（corticalsomatosensory evoked potential，CSEP）和运动诱发电位（motorevoked potential，MEP）。采用spss19.0统计分析软件包进行数据处理，所测数据均以±s表示，采用正交实验的方差分析法，α=0.05，当P＜0.05时认为差异有统计学意义。第三部分：脊柱IGRT计划中包括：放疗射野5、7、9野3水平；分割剂量16Gy、20Gy2水平；剂量率4Gy/min、6Gy/min2水平；射线角度等角度、不等角度2水平，采用L12（3×23）正交试验表对以上因素进行分配，共需12只比格犬。重复两次实验，共需36只比格犬。选用雄性成年比格犬36只，随机数字表法随机后，在5周内对其各犬胸7～12椎体行总剂量为80Gy的IGRT。并于放疗3个月后活杀取材，取各犬相同节段、相同部位（胸7～12）脊髓。采用HE染色观察脊髓神经细胞形态学的改变，透射电镜观察脊髓神经细胞超微结构改变，采用TUNEL法定量测定脊髓神经细胞凋亡情况，采用免疫组织化学法半定量测定脊髓组织中Caspase-3蛋白表达量，采用RT-PCR方法定量测定脊髓组织中Caspase-3mRNA基因表达量。采用spss19.0统计分析软件包进行数据处理，所测数据均以±s表示，采用正交实验的方差分析法，α=0.05，当P＜0.05时认为差异有统计学意义。
结果：
第一部分：①本研究所应用的方法可以较好地模拟临床的脊柱图像引导放射治疗，各组犬实验顺利，效果良好；②HE染色、透射电镜、免疫组织化学法及RT-PCR法结果均提示：放疗总剂量如果达到80Gy，则达到骨坏死的病理标准，椎体骨不再具有再生能力。放疗总剂量如果大于80Gy，则脊髓生物安全性不能得到保证。第二部分：①DWI、CSEP和MEP均可敏感地检测脊柱IGRT后脊髓的损伤。②DWI、CSEP和MEP均提示：当单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度时，脊柱IGRT的脊髓生物安全性最好。第三部分：HE染色、透射电镜、TUNEL结果、免疫组织化学法及RT-PCR法检测Caspase-3蛋白及mRNA表达结果均提示：当单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度时，脊柱IGRT的脊髓生物安全性最好。
结论：
第一部分：①本研究提供了一种建立犬精确脊柱图像引导放射治疗模型的方法。②通过筛选犬脊柱IGRT放疗计划，在确保脊髓生物安全性的前提下，确定80Gy是最大的放疗总剂量。第二部分：①DWI、CSEP和MEP对放射性脊髓损伤检测敏感，可以在脊柱IGRT时定期检测，以预防放射性脊髓损伤的发生。②单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度的组合是脊柱IGRT的较优方案。第三部分：①HE染色、透射电镜、TUNEL结果、免疫组织化学法及RT-PCR法检测Caspase-3蛋白及mRNA表达结果与DWI、CSEP和MEP的结果相吻合。②单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度的组合是脊柱IGRT的较优方案。
Objective:
Section One: Establishment the model of the image guided radiation therapy ofspinal in beagle, by comparing the the biological safety effect in spinal cord of differentprograms spine image guided radiation therapy in beagle, inorder to screen the highesttotal irradiation doses.Section Two: Based on the orthogonal design principle, throughthe MRI, electromyography detecting radiotherapy after spinal cord injury, this paperdiscusses the spine IGRT plan: different levels of radiation dose, radiation field,segmentation dose rate, radiation angle to the influence degree of the spinal cord injury,in order to optimize the combination the biological safety of spinal cord best radiationscheme. Section Three: Based on the orthogonal design principle, through the HEstaining and transmission electron microscope, TUNEL (terminal deoxynucleotidyltransferase mediated dUTP nick and labeling) method, immunohistochemistry methodsand RT-PCR method were used after spinal cord injury, this paper discusses the spineIGRT plan: different levels of radiation dose, radiation field, segmentation dose rate,radiation angle to the influence degree of the spinal cord injury, in order to optimize thecombination the biological safety of spinal cord best radiation scheme.
Methods:
Section One: Select purebred beagles and six were randomly divided: Six beagleswere irradiated by eight different models of IGRT, and the total irradiation doses were50Gy60Gy70Gy80Gy90Gy and100Gy. Mimic a beagle clinical model of tumor inthe seventh and twelveth thoracic vertebrae. The samples of spinal cord and vertebralbody bone tissue were taken out from the same locum of the seventh and twelveththoracic vertebrae on third month after radiation. The samples of the vertebral body bone tissue were observed by the hematoxylin and eosin stain and the electron microscop. Thesamples of the spinal cord were observed by the hematoxylin and eosin stain and theelectron microscopee, to observe the change of the spinal nerve cells, spinal vascularendothelial cell ultrastructure. Terminal deoxynucleotidyl transferase mediated dUTPnick and labeling (TUNEL) technique was used to examine the apoptotic cells in thespinal nerve cells. Section Two: Spine IGRT plan includes: radiation dose (16Gy,20Gy),radiation field (5,7,9wilds), segmentation dose rate (4Gy/min,6Gy/min), radiationangle (equal angle, unequal angle), using L12(3×23) orthogonal test table to the abovefactors on distribution, a total of12beagles needed. Repeat the experiment two times, atotal of36beagles needed. Choose36male adult beagles, random number table methodrandom, in5weeks on the each beagles chest7～12vertebral bodies for IGRT.Conventional MRI and diffusion-weighted imaging (DWI) were carried out every2weeks before radiotherapy and after began to radiotherapy. Cortical somatosensoryevoked potential (CSEP) and motorevoked potentialminimum-error-probability (MEP)were carried out before radiotherapy and radiotherapy after12hours,1day,1week,2weeks respectively.Section Three: Spine IGRT plan includes: radiation dose (16Gy,20Gy), radiation field (5,7,9wilds), segmentation dose rate (4Gy/min,6Gy/min),radiation angle (equal angle, unequal angle), using L12(3×23) orthogonal test table to theabove factors on distribution, a total of12beagles needed. Repeat the experiment twotimes, a total of36beagles needed. Choose36male adult beagles, random number tablemethod random, in5weeks on the each beagles chest7～12vertebral bodies for IGRT.The samples of spinal cord were taken out on the3th month after radiation. The sampleswere observed by the hematoxylin and eosin stain and the electron microscope. Terminaldeoxyn-ucleotidyl transferase mediated dUTP nick and labeling (TUNEL) technique wasused to examine the apoptotic cells in the spinal cord. The expression of Caspase-3andCaspase-3mRNA were evaluated in spine by immunohistochemistry and RT-PCR.
Results:
Section One:①This research application method can simulate the spine clinicalimage-guided radiation therapy, the dog experiment smoothly, and good effect.②Theresults of the the hematoxylin and eosin stain and the electron microscope, and theexpression of Caspase-3and Caspase-3mRNA evaluated in spine byimmunohistochemistry and RT-PCRprompted that: if the total radiation dose was80Gy,then achieved osteonecrosis pathological standard, vertebral body bone regeneration were no longer. The total radiation dose if more than80Gy, the spinal cord biological safetycouldnot be guaranteed. Section Two:①DWI, CSEP and MEP can sensitively detectspinal cord injury after spine IGRT.②DWI, CSEP and MEP suggested: when radiationdose was16Gy, segmentation dose rate was4Gy/min, radiation field was9wilds andradiation angle was equal angle, the biological safety of the spinal cord was thebest.Section Three:①The results of the the hematoxylin and eosin stain and the electronmicroscope, the terminal deoxynucleotidyl transferase mediated dUTP nick and labeling(TUNEL) technique, and the expression of Caspase-3and Caspase-3mRNA evaluated inspine by immunohistochemistry and RT-PCR prompted that: when radiation dose was16Gy, segmentation dose rate was4Gy/min, radiation field was9wilds and radiationangle was equal angle, the biological safety of the spinal cord was the best.
Conclusions:
Section One:①Research provides a beagle accurate spine image-guided radiationtherapy model establishment method.②Through the screening beagle spine IGRTradiotherapy planning, to ensure the safety of the spinal cord in biological premise, the80Gy is the biggest total dose radiation therapy. Section Two:①DWI, CSEP and MEPcan sensitively detect spinal cord injury after spine IGRT. DWI, CSEP and MEP could bedetected in the spine IGRT periodically to prevent the occurrence of radioactive spinalcord damage occurs.②The better spine IGRT program is radiation dose of16Gy,segmentation dose rate of4Gy/min, radiation field of9wilds and radiation angle of equalangle.Section Three:①HE staining and transmission electron microscope, TUNELresults, immune histochemical method and RT-PCR method to detect Caspase3proteinand mRNA expression results and DWI, is the result of MEP and CSEP can match.②The better spine IGRT program is radiation dose of16Gy, segmentation dose rate of4Gy/min, radiation field of9wilds and radiation angle of equal angle.
第一部分：建立犬精确脊柱图像引导放射治疗（Image guided radiation therapy，IGRT）模型，筛选脊柱IGRT在确保脊髓生物安全性的前提下，最大的放疗总剂量。第二部分：基于正交设计原理，通过MRI、肌电图检测放疗后脊髓损伤，探讨脊柱IGRT计划中的：不同水平的放疗射野、分割剂量、剂量率、射线角度对脊髓损伤的影响程度，以优化组合出脊髓生物安全性最好的放疗方案。第三部分：基于正交设计原理，通过HE染色、透射电镜、TUNEL法、免疫组织化学法及RT-PCR法检测放疗后脊髓损伤，探讨脊柱IGRT计划中的：不同水平的放疗射野、分割剂量、剂量率、射线角度对脊髓损伤的影响程度，以优化组合出脊髓生物安全性最好的放疗方案。
方法：
第一部分：选用雄性成年比格犬6只，随机数字表法随机分组，模拟犬胸7～12椎体及附件转移瘤，以IGRT方式分别对6只比格犬胸7～12椎体及附件给予50、60、70、80、90和100Gy剂量的放疗，于放疗3个月后活杀取材，取相同部位、相同位置的胸7～12节段椎体骨组织、脊髓材料。采用HE染色观察不同放疗剂量组骨组织的形态学的改变，透射电镜观察不同放疗剂量骨细胞超微结构改变。采用HE染色观察不同放疗剂量组脊髓神经细胞形态学的改变，透射电镜观察脊髓神经元细胞、脊髓神经髓鞘超微结构改变，采用TUNEL法观察不同放疗剂量组脊髓神经元细胞凋亡情况。第二部分：脊柱IGRT计划中包括：放疗射野5、7、9野3水平；分割剂量16Gy、20Gy2水平；剂量率4Gy/min、6Gy/min2水平；射线角度等角度、不等角度2水平，采用L12（3×23）正交试验表对以上因素进行分配，共需12只比格犬。重复两次实验，共需36只比格犬。选用雄性成年比格犬36只，随机数字表法随机后，在5周内对其各犬胸7～12椎体行总剂量为80Gy的IGRT。并于放疗前、放疗开始后每2周进行常规MRI及弥散加权成像（diffusion-weighted imaging，DWI）检测；于放疗前、放疗后12小时、1天、1周、2周分别检测皮质体感诱发电位（corticalsomatosensory evoked potential，CSEP）和运动诱发电位（motorevoked potential，MEP）。采用spss19.0统计分析软件包进行数据处理，所测数据均以±s表示，采用正交实验的方差分析法，α=0.05，当P＜0.05时认为差异有统计学意义。第三部分：脊柱IGRT计划中包括：放疗射野5、7、9野3水平；分割剂量16Gy、20Gy2水平；剂量率4Gy/min、6Gy/min2水平；射线角度等角度、不等角度2水平，采用L12（3×23）正交试验表对以上因素进行分配，共需12只比格犬。重复两次实验，共需36只比格犬。选用雄性成年比格犬36只，随机数字表法随机后，在5周内对其各犬胸7～12椎体行总剂量为80Gy的IGRT。并于放疗3个月后活杀取材，取各犬相同节段、相同部位（胸7～12）脊髓。采用HE染色观察脊髓神经细胞形态学的改变，透射电镜观察脊髓神经细胞超微结构改变，采用TUNEL法定量测定脊髓神经细胞凋亡情况，采用免疫组织化学法半定量测定脊髓组织中Caspase-3蛋白表达量，采用RT-PCR方法定量测定脊髓组织中Caspase-3mRNA基因表达量。采用spss19.0统计分析软件包进行数据处理，所测数据均以±s表示，采用正交实验的方差分析法，α=0.05，当P＜0.05时认为差异有统计学意义。
结果：
第一部分：①本研究所应用的方法可以较好地模拟临床的脊柱图像引导放射治疗，各组犬实验顺利，效果良好；②HE染色、透射电镜、免疫组织化学法及RT-PCR法结果均提示：放疗总剂量如果达到80Gy，则达到骨坏死的病理标准，椎体骨不再具有再生能力。放疗总剂量如果大于80Gy，则脊髓生物安全性不能得到保证。第二部分：①DWI、CSEP和MEP均可敏感地检测脊柱IGRT后脊髓的损伤。②DWI、CSEP和MEP均提示：当单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度时，脊柱IGRT的脊髓生物安全性最好。第三部分：HE染色、透射电镜、TUNEL结果、免疫组织化学法及RT-PCR法检测Caspase-3蛋白及mRNA表达结果均提示：当单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度时，脊柱IGRT的脊髓生物安全性最好。
结论：
第一部分：①本研究提供了一种建立犬精确脊柱图像引导放射治疗模型的方法。②通过筛选犬脊柱IGRT放疗计划，在确保脊髓生物安全性的前提下，确定80Gy是最大的放疗总剂量。第二部分：①DWI、CSEP和MEP对放射性脊髓损伤检测敏感，可以在脊柱IGRT时定期检测，以预防放射性脊髓损伤的发生。②单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度的组合是脊柱IGRT的较优方案。第三部分：①HE染色、透射电镜、TUNEL结果、免疫组织化学法及RT-PCR法检测Caspase-3蛋白及mRNA表达结果与DWI、CSEP和MEP的结果相吻合。②单次放疗剂量为16Gy，剂量率为4Gy/min、射野数为9野及射线角度为等角度的组合是脊柱IGRT的较优方案。
Objective:
Section One: Establishment the model of the image guided radiation therapy ofspinal in beagle, by comparing the the biological safety effect in spinal cord of differentprograms spine image guided radiation therapy in beagle, inorder to screen the highesttotal irradiation doses.Section Two: Based on the orthogonal design principle, throughthe MRI, electromyography detecting radiotherapy after spinal cord injury, this paperdiscusses the spine IGRT plan: different levels of radiation dose, radiation field,segmentation dose rate, radiation angle to the influence degree of the spinal cord injury,in order to optimize the combination the biological safety of spinal cord best radiationscheme. Section Three: Based on the orthogonal design principle, through the HEstaining and transmission electron microscope, TUNEL (terminal deoxynucleotidyltransferase mediated dUTP nick and labeling) method, immunohistochemistry methodsand RT-PCR method were used after spinal cord injury, this paper discusses the spineIGRT plan: different levels of radiation dose, radiation field, segmentation dose rate,radiation angle to the influence degree of the spinal cord injury, in order to optimize thecombination the biological safety of spinal cord best radiation scheme.
Methods:
Section One: Select purebred beagles and six were randomly divided: Six beagleswere irradiated by eight different models of IGRT, and the total irradiation doses were50Gy60Gy70Gy80Gy90Gy and100Gy. Mimic a beagle clinical model of tumor inthe seventh and twelveth thoracic vertebrae. The samples of spinal cord and vertebralbody bone tissue were taken out from the same locum of the seventh and twelveththoracic vertebrae on third month after radiation. The samples of the vertebral body bone tissue were observed by the hematoxylin and eosin stain and the electron microscop. Thesamples of the spinal cord were observed by the hematoxylin and eosin stain and theelectron microscopee, to observe the change of the spinal nerve cells, spinal vascularendothelial cell ultrastructure. Terminal deoxynucleotidyl transferase mediated dUTPnick and labeling (TUNEL) technique was used to examine the apoptotic cells in thespinal nerve cells. Section Two: Spine IGRT plan includes: radiation dose (16Gy,20Gy),radiation field (5,7,9wilds), segmentation dose rate (4Gy/min,6Gy/min), radiationangle (equal angle, unequal angle), using L12(3×23) orthogonal test table to the abovefactors on distribution, a total of12beagles needed. Repeat the experiment two times, atotal of36beagles needed. Choose36male adult beagles, random number table methodrandom, in5weeks on the each beagles chest7～12vertebral bodies for IGRT.Conventional MRI and diffusion-weighted imaging (DWI) were carried out every2weeks before radiotherapy and after began to radiotherapy. Cortical somatosensoryevoked potential (CSEP) and motorevoked potentialminimum-error-probability (MEP)were carried out before radiotherapy and radiotherapy after12hours,1day,1week,2weeks respectively.Section Three: Spine IGRT plan includes: radiation dose (16Gy,20Gy), radiation field (5,7,9wilds), segmentation dose rate (4Gy/min,6Gy/min),radiation angle (equal angle, unequal angle), using L12(3×23) orthogonal test table to theabove factors on distribution, a total of12beagles needed. Repeat the experiment twotimes, a total of36beagles needed. Choose36male adult beagles, random number tablemethod random, in5weeks on the each beagles chest7～12vertebral bodies for IGRT.The samples of spinal cord were taken out on the3th month after radiation. The sampleswere observed by the hematoxylin and eosin stain and the electron microscope. Terminaldeoxyn-ucleotidyl transferase mediated dUTP nick and labeling (TUNEL) technique wasused to examine the apoptotic cells in the spinal cord. The expression of Caspase-3andCaspase-3mRNA were evaluated in spine by immunohistochemistry and RT-PCR.
Results:
Section One:①This research application method can simulate the spine clinicalimage-guided radiation therapy, the dog experiment smoothly, and good effect.②Theresults of the the hematoxylin and eosin stain and the electron microscope, and theexpression of Caspase-3and Caspase-3mRNA evaluated in spine byimmunohistochemistry and RT-PCRprompted that: if the total radiation dose was80Gy,then achieved osteonecrosis pathological standard, vertebral body bone regeneration were no longer. The total radiation dose if more than80Gy, the spinal cord biological safetycouldnot be guaranteed. Section Two:①DWI, CSEP and MEP can sensitively detectspinal cord injury after spine IGRT.②DWI, CSEP and MEP suggested: when radiationdose was16Gy, segmentation dose rate was4Gy/min, radiation field was9wilds andradiation angle was equal angle, the biological safety of the spinal cord was thebest.Section Three:①The results of the the hematoxylin and eosin stain and the electronmicroscope, the terminal deoxynucleotidyl transferase mediated dUTP nick and labeling(TUNEL) technique, and the expression of Caspase-3and Caspase-3mRNA evaluated inspine by immunohistochemistry and RT-PCR prompted that: when radiation dose was16Gy, segmentation dose rate was4Gy/min, radiation field was9wilds and radiationangle was equal angle, the biological safety of the spinal cord was the best.
Conclusions:
Section One:①Research provides a beagle accurate spine image-guided radiationtherapy model establishment method.②Through the screening beagle spine IGRTradiotherapy planning, to ensure the safety of the spinal cord in biological premise, the80Gy is the biggest total dose radiation therapy. Section Two:①DWI, CSEP and MEPcan sensitively detect spinal cord injury after spine IGRT. DWI, CSEP and MEP could bedetected in the spine IGRT periodically to prevent the occurrence of radioactive spinalcord damage occurs.②The better spine IGRT program is radiation dose of16Gy,segmentation dose rate of4Gy/min, radiation field of9wilds and radiation angle of equalangle.Section Three:①HE staining and transmission electron microscope, TUNELresults, immune histochemical method and RT-PCR method to detect Caspase3proteinand mRNA expression results and DWI, is the result of MEP and CSEP can match.②The better spine IGRT program is radiation dose of16Gy, segmentation dose rate of4Gy/min, radiation field of9wilds and radiation angle of equal angle.
引文
[1]李晔,赵宏,王以朋.脊柱转移瘤的临床治疗进展[J].中华医学杂志,2009,89(33):2372-2374.
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