【作者】 李凯；

【导师】 王娟；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2007， 硕士

【摘要】 目的:奥沙利铂属于第三代铂类制剂,在结直肠癌、胃癌等肿瘤化学治疗中占有重要地位。其临床剂量限制毒性为周围神经毒性。其引起的周围神经病严重时可影响肢体功能导致患者的生活质量下降,而且还有可能使化学治疗中断。因此寻找有效预防和治疗其神经毒性的药物成为人们的焦点。还原型谷胱甘肽是一种有希望对其周围神经毒性产生保护作用的药物。但在其预防顺铂临床神经毒性的研究中,尚存在争议。国内外GSH保护奥沙利铂致周围神经毒性的研究鲜有报道,本实验旨在从细胞形态学及神经生物学方面研究奥沙利铂致大鼠周围神经系统毒性损害是否有可恢复的趋势;并初步研究还原型谷胱甘肽对奥沙利铂致大鼠周围神经毒性的保护作用。方法:健康雄性Wistar大鼠120只,随机分成对照组、奥沙利铂组、奥沙利铂加还原型谷胱甘肽组,每组40只;对照组大鼠腹腔注射5%葡萄糖30ml/kg,奥沙利铂组和奥沙利铂加GSH组腹腔注射奥沙利铂20mg/kg+5%葡萄糖30ml/kg ,奥沙利铂20mg/kg+GSH500mg/kg+5 %葡萄糖30ml/kg,每组再随机据10个时间点分为:0小时、24小时、48小时、1、2、3、4、5、6、7周分为10个亚组。每天观察大鼠的一般情况;均在上述时间点灌注后取大鼠左侧L4-5后根神经节。进行石蜡切片制作,HE染色、免疫组织化学染色,光镜观察雄性大鼠后根神经节内的感觉神经元细胞的损伤情况,并利用图像分析系统对神经元胞体、胞核、以及核仁的形态学变化以及神经生长因子免疫阳性细胞的平均吸光度进行测定,对所得的数据利用GraphPad Prism Version 4.0进行统计学分析。结果:1.对一般毒性的保护:本实验中加GSH组大鼠行为迟缓、觅食减少、腹泻、皮肤黏膜出血点的发生率较奥沙利铂组大鼠明显减低。用药1-7周,奥沙利铂组大鼠体重与对照组相比有明显的统计学差异(P<0.0001);加GSH组大鼠体重与对照组相比,在第1、2周有统计学上的差异(P<0.05),3周后与对照组相比无统计学上的差异(P>0.05);用药第1-7周,加GSH组的体重与奥沙利铂组相比都有统计学的差异(P<0.05)。2.细胞形态学观察:奥沙利铂组损伤的主要靶器官为大鼠后根神经节感觉神经元,并以神经元细胞核仁改变较为显著,细胞体、细胞核以及核仁均可见不同程度的固缩现象,细胞质也有浓缩现象。亦可见部分神经元胞体缩小,胞浆浓缩,与周围细胞出现分离;同时亦有部分胞核染色变浅,核膜轮廓模糊;核仁碎裂比例、多细胞核现象有所增高;其中以第2天核仁变化最明显,第1周后形态学改变逐渐改善,24小时到6周的胞体﹑胞核及核仁面积与对照组比较差异有统计学意义(P<0.05);7周左右基本恢复正常。奥沙利铂加GSH组大鼠后跟神经节神经元第1、2天与第1周胞体﹑胞核及核仁面积与奥沙利铂组比较差异无统计学意义(P>0.05);2-7周差异有统计学意义(P<0.05);奥沙利铂加GSH组大鼠后跟神经节神经元第1、2天与第1、2周胞体﹑胞核及核仁面积与对照组比较差异有统计学意义(P<0.05);3周后与对照组比较差异无统计学意义(P>0.05)。3.对神经生长因子表达影响:奥沙利铂组大鼠后跟神经节内NGF表达呈现下调-回升趋势,即在用药后第1、2天表达下调最为显著,6周后基本恢复正常,具有时间依赖性的特点,而奥沙利铂加GSH组神经节内NGF表达也呈下调-回升趋势,但与奥沙利铂组相比明显好转即在第1周就恢复到正常水平,与对照组相比无统计学差异(P>0.05)。结论:GSH减轻奥沙利铂致大鼠的毒性,大鼠的一般毒性随着时间的延长呈现可恢复的趋势但需要时间较长,加用GSH可使其恢复的时间缩短;GSH加奥沙利铂组大鼠周围神经系统的异常病理学改变均较奥沙利铂组轻微;且大鼠神经元内NGF的表达较奥沙利铂组明显增高。更多还原

【Abstract】 Objective: Oxaliplatin is a third-generation platinum-based chemotherapeutic agent and play an important role in the colorectal cancer and gartric carcinoma treatment. Neurotoxicity is its dose-limiting symptom. In humans, two different types of neurological symptoms occur during oxaliplatin treatment. Disabling peripheral neuropathy has a significant negative impact on quality of life and interrupt the chemotherapy. Accordingly, potential neuroprotective agents are to be investigated. Glutathione is a hopeful chemoprotectant to ameliorate the toxicity associated with platinum-based chemotherapeutic agent. But there exist objection whether or not neuroprotective effect of GSH in clinical neurotoxicity of cisplatin. There have been few studies that neuroprotective effect of GSH in experiment of neurotoxicity of oxalplatin. The aim of this study was to investigate whether the pathological changes of peripheral neurotoxicity induced by oxlaiplatin depended on recovery time. This experiment also initially investigate the potential neuroprotective effect of GSH in dorsal root ganglion neurons in vivo from pathology and neurobiology . Methods: 120 healthy male Wistar rats were randomly divided into three groups (n=40 in each): control group were given 5%glucose 30ml/kg and study group were given single intraperitoneal doses of oxaliplatin20mg/kg and in combination with reduced glutathione 500mg/kg. These three groups were also divided into 10 subgroups respectively. These 10 groups represented 10 time points from 0 hours、24 hours、48 hours、1、2、3、4、5、6 to 7 weeks. Left L4-5 dorsal root ganglia and sciatic nerves of all rats were dissected out at different points. DRG sensory neurons and sciatic nerves of all rats were studied by the light microscopy and transmission electron microscopy technique to define the pathological changes at various time points when treated with a single dose of oxaliplatin and a single dose of GSH before intraperitioneal oxaliplatin. The expression of NGF in DRG was detected by immunohistochemistry. DRG sensory neurons and the expression of NGF were analyzed by Digital Medical Image Analysis System to determine the morphologhical changes of DRG and the expression of NGF.Results: 1. Protection of the general toxicity of oxaliplatin. GSH group of slowly acts, reduction foraging, diarrhea, bleeding of the skin mucosa general toxicity rates significantly lower than oxaliplatin group. Medication 1-7weeks, The weight of after treated rats ,compared with the control group, has a statistically significant difference in 1,2,3,4,5,6,7 weeks(P<0.0001).Oxaliplatin plus GSH compared with the control group has significant difference in 1,2-week( P <0.05); and has significant difference compared with the group of rats were given single intraperitoneal doses of oxaliplatin(P<0.05)in 1-7 weeks. 2. The results of neuron morphology. In the period of oxaliplatin treatment, DRG was the main target of the peripheral neurotoxicity. The nucleolus showed the most prominent pathological changes. The neuropathological examination evidenced shrinkage of DRG soma, nucleus, and nucleolus were shown in the treated rats. All these changes were evident at 48 hours and almost completely recovered in 7 weeks. All these changes of oxaliplatin plus GSH in rats minor oxaliplatin group in 24-48hours, neuronal cell bodies in the cytoplasm and nuclei and nucleoli area was statistically significant differences with the control group( P <0.05). The morphological changes gradually improve after the first week. The area of cellular, nucleus and nucleoli was statistically significant difference compared with the control group (P<0.05). It returned to be normal in 7 weeks. The area of cellular, nucleus and nucleoli of GSH plus oxaliplatin group in rats was no significant difference compared with the oxaliplatin group in 1,2days and 1 week, and the difference was statistically significant compared with the control group (P<0.05); After two weeks was no significant difference compared with the oxaliplatin group (P>0.05) 3. The effect of NGF expression: The NGF expression was evident at 24-48hours and completely recovered in 6 weeks. GSH plus oxaliplatin group was down-rising trend, however, there was no significant difference between the control group after 1 weeks(P>0.05).Conclusion: GSH reduces the toxicity induced by oxaliplatin. The toxicity showed a recovery trend and need long time. GSH maybe reduce the oxaliplatin-induced abnormal of changes of neurons of DRG. NGF expression of plus GSH was markedly higher than the control group was not protected.更多还原