【作者】 崔宁；

【导师】 谢克勤；

【作者基本信息】 山东大学 ， 毒理学， 2005， 硕士

【摘要】 目的 2,5-己二酮(HD)是重要的工业有机溶剂正己烷的活性代谢产物,介导了正己烷诱导的神经退行性病变,其具体的发病机制尚不清楚。本研究通过建立大鼠HD亚慢性中毒模型,测定神经组织中关键的凋亡调控蛋白Bax、Bcl-2和Caspase-3含量变化,对中枢神经细胞进行凋亡形态学和生化检测,研究细胞凋亡机制是否参与了HD诱导的神经退行性变,为进一步探讨HD的神经毒性机制提供依据。 方法 Wistar雄性成年大鼠60只随机分为对照组、低剂量组和高剂量组,分别腹腔注射生理盐水、200mg HD/kg体重和400mg HD/kg体重,每周5次,连续2个月。每周观察大鼠中毒表现,测量体重、热觉传导、压痛阈值、后肢撑力、转棒实验等指标。采用Western blotting方法分析各实验组大脑、小脑、脊髓和坐骨神经中Bax、Bcl-2和Caspase-3含量变化,用免疫组织化学方法原位检测大脑、小脑和脊髓组织中的Bax和Bcl-2蛋白的表达,并用苏木素-伊红染色(HE)和TUNEL方法分析大脑、小脑和脊髓组织中神经细胞的形态和DNA改变。 结果 一、体重及神经功能测试 HD染毒后大鼠体重显著降低,并且随剂量的升高,呈现一定的剂量-效应关系。至实验结束,与对照组相比,低剂量组体重降低了20%(P<0.01),高剂量组体重降低了43%(P<0.01)。与对照组相比,高剂量组在热板仪上的保持时间有延长的趋势(延长了23%),但无显著性差异(P>0.05),高剂量组压痛阈值显著增更多还原

【Abstract】 Objectives 2,5-hexanedione (HD), as an active metabolite of industrial organic solvent n-hexane, is believed to mediate the neurodegeneration which ensues as a result of n-hexane exposure. The exact molecular mechanism of HD-induced neurodegeneration remains far from elucidated. In this study, we examined the alterations of three apoptosis-regulatory proteins Bcl-2, Bax and Caspase-3 in nerve tissues and detect the characteristic morphological changes and DNA cleavage in central neurons in rats subchronically exposed to HD, in order to determine the real role of apoptosis in this disorder.Methods 60 adult male Wistar rats were randomly divided into three groups: control group, lower-dose rate group and higher-dose rate group. HD was injected ip daily (5 days per week for 2 months) to assigned groups of rats according to two dosing rates i.e., 200 and 400mg/kg/day. And Age-matched control rats received an equivalent volume of saline by ip. Body weights and the developments of intoxicated symptoms including the observations of spontaneous locomotion in an open field, measurements of foot splay in hindlimb landing, nervous sensory to pain and heat in hindlimbs and the rotarod test were measured once a week. The alterations of Bax, Bcl-2 and Caspase-3 proteins in cerebrum, cerebellum, spinal cord and sciatic nerve were detected by Western blotting. What’s more, the expressions of Bax and Bcl-2 in cerebellum, cerebellum and spinal cord were tested in situ by immunohistochemistry. HE and TUNEL were also used to investigate the morphological changes and DNA cleavages in central neuron cells.Results1 .Body weights and neurobehavioral functions tests: The body weights of rats decreased dramatically after HD exposure, exhibiting a dose-effect relationship. At the end, the body weights decreased by 20%(P<0.01) in lower-dose rate group and by 43%(/><0.01) in higher-dose rate group compared with control group. In higher-dose rate group, though the threshold of sensory nerve to heat increased by 23%, the change had no significant difference compared with control group. Meanwhile, the threshold of sensory nerve to press pain in higher-dose rate group remarkably increased by 18%(P<0.05). Changes of the two thresholds in lower-dose rate group did not make sense. The hindlimb landing foot splay in higher-dose rate group increased by 46%(P<0.01) at the forth week of HD exposure and this index in lower-dose rate group increased by 15%(P<0.05) at the sixth week of exposure. The index of rotarod test in higher-dose rate group reduced by 62%(P<0.01) and by 49%(P<0.05) in lower-dose rate group, as compared to control.2. Western blotting analysis indicated that the expressions of Bax, Bcl-2 and Caspase-3 were elevated significantly in all nervous tissues except in spinal cord, which the changes were too little to make sense.In cerebrum, levels of Bax increased by 48% in lower-dose rate group (PO.OI) and by 65% in higher-dose rate group (P<0.01); levels of Bcl-2 increased by 30% in lower-dose rate group (P<0.01) and by 52% in higher-dose rate group (PO.01); meanwhile, the expression of Caspase-3 was elevated by 30% in lower-dose rate group (PO.05) and by 78% in higher-dose rate group (PO.01).In cerebellum, expressions of the three proteins were all significantly elevated in higher-dose rate group, expressions of Bax, Bcl-2 and Caspase-3 increased by 33%(PO.05), by 39%(PO.01) and by 182%(P<0.01), respectively. In lower-dose rate group, expression of Caspase-3 was elevated by 126%(P<0.05), while expressions of the other two proteins did not change significantly.In spinal cord, the changes of expressions of Bax, Bcl-2 and Caspase-3 were too little to make sense, compared to control group.In sciatic nerves, the extent of Bax expression increased by 67%(P<0.01) in lower-dose rate group and by 79%(P<0.01) in higher-dose rate group, compared to the control group. The expression of Bcl-2 was elevated by 41%(P<0.01) in lower-dose rate group and by 27%(P<0.01) in higher-dose rate group. The expression of Caspase-3 was elevated by 44%(/><0.05) in lower-dose rate group and by 283%(P<0.01) in higher-dose rate group.3. Immunohistochemistry analysis exhibited that expressions of Bax and Bcl-2 in central neuron cells significantly increased, after the exposure to HD.In cerebrum, in comparison to control group, the expression of Bax increased by 20%(P<0.05) in lower-dose rate group and by 41%(P<0.05) in higher-dose rate group. The expression of Bcl-2 increased by 24%(/)<0.05) in lower-dose rate group and by 91%(P<0.01) in higher-dose rate group.In cerebellum, the change of expression of Bax in lower-dose rate group did not make sense, while the expression of Bcl-2 in lower-dose rate group was elevated by 43%(P<0.01). In higher-dose rate group, the expression of Bax and Bcl-2 was elevated by 24%(/><0.01) and by 62%(P<0.01), respectively.In spinal cord, in comparison to control group, the expression of Bax increased by 67%(P<0.01) in lower-dose rate group and by 33%(P<0.05) in higher-dose rate group. The expression of Bcl-2 increased by 36%(/><0.05) in lower-dose rate group and by 31 %(P<0.05) in higher-dose rate group.4. HE and TUNEL analysis indicated that the central neuron cells did not exhibit remarkable morphologic changes like apoptosis, nor were TUNEL positive cells detected, suggesting no apparent DNA cleavages.Conclusions1. HD can significantly affect the sensory and motor functions of nervous system, providing evidence for the neurotoxicity of HD.2. It was firstly proved that the expressions of Bax, Bcl-2 and Caspase-3 proteins in central and peripheral nerve tissues are up-regulated. The changes of Caspase-3 are the most significant of the three proteins.3. The alterations of Bax, Bcl-2 and Caspase-3 in sciatic nerves are the most significant in all nerve tissues detected.4. Morphologically, central nervous cells do not exhibit apparent changes like apoptosis, nor the DNA cleavages.5. It is the first time that apoptosis-regulatory proteins Bax, Bcl-2 and Caspase-3 are found to participate in HD induced neurodegeneration, however, the role of apoptosis in the mechanism of HD neurotoxicity still requires further study.更多还原