【作者】 陈宏；

【导师】 李红戈； 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2008， 硕士

【摘要】 第一部分重组人促红细胞生成素预处理对黑质多巴胺神经元生存预后的影响目的:研究重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)预处理对帕金森病模型中黑质多巴胺神经元生存预后的影响。方法:健康SD大鼠分为五组:空白对照组,6-羟多巴组(6-Hydroxydopamine,6-OHDA), 6-OHDA +1u/ml rhEPO组,6-OHDA +6u/ml rhEPO组,6-OHDA +10u/ml rhEPO组。6-OHDA浸泡大鼠黑质脑片建立离体帕金森病模型。不同浓度rhEPO在不同时间点预处理黑质多巴胺神经元,观察黑质中酪氨酸羟化酶(tyrosine hydroxylase,TH)变化。结果:rhEPO预处理能抑制6-羟多巴导致的黑质酪氨酸羟化酶免疫反应(tyrosine hydroxylase–immunoreactive,TH–IR)阳性细胞数减少,6u/ml rhEPO预处理在3h与6h时间点的保护作用优于1u/ml rhEPO预处理(P<0.01, P<0.05),10u/ml rhEPO预处理在3h与6h时间点的保护作用也优于1u/ml rhEPO预处理;但6u/ml rhEPO预处理与10u/ml rhEPO预处理对多巴胺神经元的保护作用差别无统计学意义(P>0.05)。结论:rhEPO预处理对黑质多巴胺神经元具有保护作用。第二部分重组人促红细胞生成素预处理对6-羟多巴诱导的离体帕金森病模型黑质多巴胺神经元凋亡的影响目的:上一部分的实验表明了重组人促红细胞生成素(recombinant human erythropoietin ,rhEPO)对离体帕金森病模型中黑质多巴胺神经元具有保护作用,本部分研究rhEPO预处理对离体帕金森病模型中黑质多巴胺神经元保护作用的机制。方法:18只SD大鼠分成3组,脑片分别位于空白组、6-羟多巴组、6-羟多巴+6u/ml rhEPO组的人工脑脊液II中孵育6小时。然后用免疫组化方法观测黑质中Caspase-3免疫反应阳性细胞数的变化,TUNEL法观测黑质中多巴胺神经元的凋亡情况。结果:与6-OHDA组相比,6-OHDA+6u/ml rhEPO组中的Caspase-3免疫反应阳性细胞及凋亡细胞减少,差异有统计学意义(P<0.01, P<0.01)。结论:rhEPO预处理减轻6-OHDA对离体帕金森病模型中多巴胺神经元的损伤,其机制可能与rhEPO抑制黑质多巴胺神经元凋亡有关。第三部分重组人促红细胞生成素预处理对6-羟多巴诱导的离体帕金森病模型黑质多巴胺神经元氧化应激的影响目的:第一部分的实验表明了重组人促红细胞生成素(recombinant human erythropoietin ,rhEPO)对离体帕金森病模型中黑质多巴胺神经元具有保护作用,本部分试验进一步研究rhEPO预处理对离体帕金森病模型中黑质多巴胺神经元保护作用的机制。方法:6-羟多巴浸泡大鼠黑质脑片建立离体帕金森病模型。用6u/ml rhEPO预处理黑质多巴胺神经元,观察黑质氧化应激指标—丙二醛、谷胱甘肽、超氧化物歧化酶和谷胱甘肽过氧化物酶的变化。结果:与6-羟多巴组相比,6u/ml rhEPO预处理组的丙二醛值降低,谷胱甘肽、超氧化物歧化酶、谷胱甘肽过氧化物酶值增高。结论:rhEPO预处理对黑质多巴胺神经元的保护作用的机制可能与rhEPO减轻氧化应激反应有关。更多还原

【Abstract】 PartⅠExperimental study on effect of preconditioning with recombinant human erythropoietin on prognosis of dopaminergic neurons in substantia nigra of rat with Parkinson’s disease induced by 6-hydroxydopamine in vitro.Object:Study the effect of preconditioning with recombinant human erythropoietin (rhEPO) on the survival of dopaminergic(DA) neurons in substantia nigra(SN) of rat with Parkinson’s disease (PD) induced by 6-hydroxydopamine(6-OHDA) in vitro. Methods: The healthy Sprague-Dawley(SD) rats were divided into 5 groups:control group, 6-OHDA group, 6-OHDA+1u/ml rhEPO group,6-OHDA+6u/ml rhEPO group, 6-OHDA+10u/ml rhEPO group. PD models in vitro were established by incubating rat midbrain slices containing SN in artificial cerebrospinal fluid II(ACSFII) containing 6-OHDA (0.05mM).The midbrain slices were preconditioned with the different concentration of rhEPO for different duration. Then observed the change of the number of tyrosine hydroxylase–immunoreactive(TH–IR) positive neurons between the different groups Results:The preconditioning with rhEPO can ameliorate the damage which was produced by 6-OHDA to DA neurons in SN of PD models in vitro. The protection of the preconditioning group with rhEPO at concentration of 6u/ml ,where the midbrain slices were incubated for 3 hours or 6 hours, is superior to the protection of the preconditioning group with rhEPO at concentration of 1u/ml where the midbrain slices were incubated for 3 hours or 6 hours ( P<0.01, P<0.05,respectively ) ;and so is the protection of the preconditioning group with rhEPO at concentration of 10u/ml ( P<0.01, P<0.05, respectively). But there is no different protection between 6u/ml rhEPO and 10u/ml rhEPO(P>0.05). Conclusion: Preconditioning with rhEPO can induce neuroprotection of DA neurons in SN of rat with PD induced by 6- OHDA in vitro. PartⅡExperimental study on effect of preconditioning with recombinant human erythropoietin on dopaminergic neuronal apoptosis in substantia nigra of rat with Parkinson’s disease induced by 6-hydroxydopamine in vitro.Object: The last experimental result have showed that the preconditioning with recombinant human erythropoietin (rhEPO)induced neuroprotection of dopaminergic neurons in substantia nigra (SN) of rat with Parkinson’s disease (PD) induced by 6-Hydroxydopamine (6-OHDA) in vitro.Here, study the mechanism of neuroprotection of preconditioning with rhEPO on dopaminergic neurons in SN of rat with PD induced by 6-OHDA in vitro. Methods:Eighteen healthy SD rats were divided into 3 groups:control group,6-OHDA group, 6-OHDA+6u/ml rhEPO group, and then the midbrain slices containing SN were incubated in artificial cerebrospinal fluid II (ACSFII) for 6 hours, respectively. Immunohistochemical technique was used to detect the expression of Caspase-3 in SN,and at the same time,TUNEL method was used to detect dopaminergic neuronal apoptosis.Results: The Caspase-3-IR positive cells and TUNEL positive cells in 6-OHDA+rhEPO group were less than those in 6-OHDA group respectively (P<0.01, P<0.01,respectively).Conclusion:Preconditioning with rhEPO can ameliorate the damage, which was produced by 6-OHDA,to DA neurons in SN of rat with PD induced by 6-OHDA in vitro,it seems likely that protection of rhEPO is associated with inhibition of DA neuronal apoptosis in SN. PartⅢExperimental study on effet of preconditioning with recombinant human erythropoietin on oxidative stress of dopaminergic neurons in substantia nigra of rat with Parkinson’s disease induced by 6-hydroxydopamine in vitroObject: The first experimental result have showed that the preconditioning with recombinant human erythropoietin (rhEPO)induced neuroprotection of dopaminergic neurons in substantia nigra (SN) of rat with Parkinson’s disease (PD) induced by 6-Hydroxydopamine (6-OHDA) in vitro.Here, study further the mechanism of neuroprotection of preconditioning with rhEPO on dopaminergic neurons in SN of rat with PD induced by 6-OHDA in vitro. Methods: PD models in vitro were established by incubating rat midbrain slices containing SN in artificial cerebrospinal fluid II(ACSFII) containing 6-OHDA (0.05mM).The midbrain slices were preconditioned with rhEPO at the concentration of 6u/ml.Then observed the change of Maleic Dialdehyde(MDA), Glutathione(GSH),Superoxide Dismutase(SOD) and Glutathione Peroxidase(GSH-PX) between the different groups. Results: The MDA decreased, and the GSH,SOD and GSH-PX increased in the preconditioning groups with rhEPO at concentration of 6u/ml when compared with those in the 6-OHDA group. Conclusion: The neuroprotection induced by preconditioning with rhEPO is possibly due to ameliorating oxidative stress.更多还原