【作者】 张玉梅；

【导师】 张万琴；

【作者基本信息】 大连医科大学 ， 生理学， 2009， 博士

【摘要】 背景与目的:帕金森病(Parkinson’s disease,PD)又称震颤麻痹(paralysis agitans),是一种中老年人常见的中枢神经系统退行性疾病,日益成为公共卫生关注的焦点。随着老龄化社会的到来,其发病率呈上升趋势。在临床上,帕金森病主要表现为运动功能障碍,如运动迟缓(slowness ofmovements)、静止性震颤(rest tremor)、僵直(rigidity)和步态异常(gaitabnormalities)等。帕金森病的特征性病理学表现是选择性黑质密质部(pars compacta of the substantia nigra,SNpc)多巴胺(dopamine,DA)能神经元进行性缺失和残存的多巴胺能神经元中出现嗜酸性路易氏小体(Lewy bodies,LB),同时纹状体多巴胺含量明显减少。帕金森病的病因至今尚未明确,根据流行病学调查、神经病理学研究及基因水平分析,认为帕金森病可能与环境因素、神经毒素、感染、遗传、年龄等因素均有关。多种因素互相作用,共同促进其发生发展。近年来,越来越多的实验证据表明泛素-蛋白酶体通路(ubiquitinproteasome pathway,UPP)功能障碍与帕金森病的发生有着密切的关系,泛素蛋白酶体通路是由泛素(ubiquitin,Ub)、泛素结合酶(ubiquitin-conjugaring enzyme,E2)、泛素激活酶(ubiquityin-activating enzyme,E1)、泛素连接酶(ubiquitin protein ligase,E3)以及26S蛋白酶体(proteasome)、去泛素化酶(deubiquitin enzymes,DUBs)共同组成。泛素蛋白酶体通路十分复杂,该通路与细胞内许多的重要生命活动联系非常密切。其中泛素是一个由76个氨基酸组成的蛋白质,分子质量为40～50kD,其内部含有7个赖氨酸残基(K6、K11、K27、K29、K33、K48和K63),这些赖氨酸残基就是多聚泛素链延长的连接位点,等待降解的蛋白质必须被多聚泛素链所标记才可能被蛋白酶体识别并降解,一旦这个过程发生问题便有可能导致异常蛋白的积聚,因此会导致帕金森病患者中脑黑质多巴胺神经元中出现路易氏小体。免疫组织化学染色结果显示此包涵体包含的主要成分是α-突触核蛋白。α-突触核蛋白就是一种在正常健康人的脑组织中广泛分布的可溶性蛋白,它是一种由140个氨基酸构成的蛋白质,主要分布于神经元的突触前终末部位,分子质量为19kD。在正常的生理条件下,α-突触核蛋白可以维护突触的正常功能进行,并参与调节多巴胺的生物合成,同时这种蛋白质还具有伴侣蛋白的功能,例如像热休克蛋白的功能,保护细胞免遭热应激和氧化应激损伤,并可以帮助细胞对抗异常蛋白聚集,促进它们的降解。当外界损伤出现时,α-突触核蛋白可能会通过聚集来抵抗损伤,随着α-突触核蛋白的不断聚集,便超出了细胞所能承受的清除能力,异常蛋白便不能被及时清除,反而对细胞产生毒性作用,加速细胞死亡。目前,PD的治疗主要分为药物对症治疗、神经保护性治疗、损伤部位修复性治疗以及基因治疗和神经外科手术治疗。迄今为止对症治疗仍然是帕金森病治疗的最主要手段。对症治疗虽然能相对减轻患者症状,但难以阻止或减缓疾病的发展,且不能保护残存的多巴胺能神经元,况且长期对症治疗会给患者带来很多并发症。因此,进一步探索帕金森病的发病机制以及明确帕金森病的预防和治疗仍然是亟待解决的问题。鉴于绝大多数帕金森病患者的发病形式为散发性,家族性帕金森病只占帕金森病患者总数的5%～10%;而且,近期研究提示,散发性帕金森病患者的核心发病机制可能是线粒体复合体Ⅰ缺陷。因此,线粒体复合体Ⅰ抑制性帕金森病动物模型应该是研究帕金森病的发病机制、尤其是探索中脑黑质多巴胺能神经元特异性死亡过程的最佳模型。鱼藤酮(rotenone)系农药老品种,是一种天然杀虫剂,在蔬菜、果树和药材等农作物的害虫防治中应用非常广泛。现在,鱼藤酮的长期使用以及慢性接触所造成的环境污染和对人类的危害日益引起关注。因为鱼藤酮具有亲脂性,可容易透过血脑屏障,对脑组织内线粒体呼吸链复合体(ComplexⅠ)即NADH脱氢酶活性具有非常强大而广泛的抑制作用,可以阻断细胞呼吸链的递氢功能以及氧化磷酸化过程,进而产生细胞毒作用。而且与其它复合体Ⅰ抑制剂不同,鱼藤酮可以直接进入多巴胺能神经元发挥它的毒性作用,而无需多巴胺转运体帮助。动物研究还发现,长期暴露于鱼藤酮的大鼠中脑黑质多巴胺能神经元内能出现典型的路易氏小体。同时,鱼藤酮给药大鼠还出现行动缓慢、僵住症等帕金森病样临床表现。因此,鱼藤酮可能是帕金森病发病的潜在因素之一。那么尽快建立稳定可靠的帕金森病鱼藤酮动物研究模型,对于探索鱼藤酮的相关作用机制,了解环境因素在帕金森病发病环节中的作用及阐明帕金森病的发病机制以及更好的预防和治疗帕金森病将具有十分重要的意义。近年来,很多研究者建立了鱼藤酮帕金森病大鼠动物模型,但均因实验条件要求太高,制作过程复杂,死亡率也高等原因难于大范围推广,因此我们实验的目的就是试图改进鱼藤酮大鼠模型的制作方法,使这一帕金森病的最佳在体研究平台易于推广,有望为帕金森病的防治研究提供更好的平台和理论依据。方法:80只SD大鼠随机分为四组:(1)梯度剂量模型组:先以2mg/kg鱼藤酮剂量给药2天,继而1mg/kg鱼藤酮剂量给药4天,最后0.5mg/kg鱼藤酮剂量给药22天,共40只大鼠;(2)恒定剂量模型组:0.5mg/kg鱼藤酮剂量给药28天,共20只大鼠;(3)模型对照组:给予同等剂量二甲基亚砜28天,共14只大鼠;(4)生理盐水对照组:给予同等剂量生理盐水28天,共6只大鼠。给药方式均采用颈背部皮下注射,每周停药2天,实验周期共计38天。每周观察一次大鼠的一般状态并称量体重,临杀前进行行为学测试。采用HPLC方法进行纹状体多巴胺及其代谢产物含量检测及中脑黑质酪氨酸羟化酶(TH)免疫组织化学染色:通过HE染色及尼氏染色方法分别观察心、肝、脾、肾等重要胸腹腔脏器及海马、顶叶皮质的形态学变化,同时进行中脑黑质和酪氨酸羟化酶免疫组织化学染色加尼氏染色:通过Western blot方法对中脑黑质和纹状体泛素蛋白(ubiquitin)及α-突触核蛋白(α-synuclein)进行含量检测,同时分别进行TH/ubiquitin和TH/α-synuclein免疫荧光化学双染。结果:1、梯度剂量模型组大鼠一般状态差,体重逐日减轻,出现明显行为学障碍:HPLC检测结果显示该组大鼠纹状体多巴胺较正常组大鼠下降达80%以上,且多巴胺代谢产物也不同程度下降,大鼠中脑黑质酪氨酸羟化酶免疫染色结果也表明梯度剂量模型组大鼠黑质致密部阳性神经元和神经纤维数目明显减少。恒定剂量模型组大鼠无行为学改变,纹状体多巴胺及其代谢产物含量正常,但中脑黑质多巴胺神经元出现损伤,程度轻于梯度剂量模型组。2、大脑顶叶皮质和海马尼氏染色及胸腹腔重要脏器HE染色结果表明梯度和恒定剂量模型组大鼠未出现相应部位损伤,中脑黑质酪氨酸羟化酶免疫染色加尼氏染色结果显示组间中脑黑质致密区以外部位尼氏小体数目无差异。3、Western-blot检测中脑黑质和纹状体α-synuclein/ubiquitin结果显示:在中脑黑质部位,梯度剂量模型组与恒定剂量模型组总α-synuclein表达均增加,以梯度剂量模型组增加明显,且在梯度剂量模型组和恒定剂量模型组,除19kD位置出现阳性条带外,在大约30kD和50kD处也出现阳性条带,纹状体部位组间表达无差异。在中脑黑质部位,梯度剂量模型组与恒定剂量模型组ubiquitin及ubiquitin化蛋白表达增加,且梯度剂量模型组增加明显,纹状体部位组间比较无差异。酪氨酸羟化酶与α-synuclein/ubiquitin免疫荧光双染结果同Western-blot检测一致。结论:1、采用颈部皮下长期低剂量注射鱼藤酮的方法,成功建立了鱼藤酮大鼠帕金森病模型,动物出现震颤、强直、活动减少等类帕金森病症状,中脑黑质多巴胺神经元严重受损,纹状体多巴胺及其代谢产物含量明显减少。2、降低鱼藤酮给药剂量,能明显减轻大鼠中脑黑质多巴胺神经元损伤程度,改善动物行为学表现,提高纹状体多巴胺及其代谢产物含量,该给药方式可以成功制备帕金森病临床前模型,亦说明鱼藤酮对多巴胺神经元的损伤作用呈现剂量依赖性。3、可以诱导帕金森病症状出现的鱼藤酮剂量长期颈部皮下注射未对大鼠海马、顶叶皮质造成病理学可见的损伤,亦未引起大鼠心、肝、脾、肾出现明显的病理学改变。长期低剂量颈部皮下注射鱼藤酮能选择性诱导中脑黑质多巴胺神经元损伤,说明中脑黑质多巴胺神经元对鱼藤酮具有高度敏感性。4、长期低剂量颈部皮下注射鱼藤酮造成多巴胺神经元损伤的大鼠其中脑黑质α-synuclein表达总量增加,并出现α-synuclein向残存多巴胺神经元内聚集,进一步证实α-synuclein聚集与多巴胺神经元损伤有关。5、长期低剂量颈部皮下注射鱼藤酮造成多巴胺神经元损伤的大鼠其中脑黑质ubiquitin及ubiquitin化蛋白表达增加,说明泛素蛋白酶体系统功能障碍与多巴胺神经元损伤有关。更多还原

【Abstract】 Background and objective:Parkinson disease(PD) is a progressive neurological disorder with a prevalence of 1-2%in people over the age of 50.It has a world-wide distribution and has no gender preference.PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of intracytoplasmatic inclusions known as Lewy bodies.The selective degeneration of the nigrostriatal dopaminergic pathway is a fundamental characteristic in Parkinson disease(PD). Although the etiology of PD remains unclear,several lines of evidence indicate that living in a rural environment,farming,drinking well water and occupational exposure to agricultural chemicals are risk factors for developing PD.Considerable evidence suggests a multifactorial etiology involving genetic and environmental factors.In recent years,an increasing number of experimental evidence shows that the ubiquitin-proteasome pathway(ubiquitin proteasome pathway, UPP) dysfunction and the occurrence of Parkinson’s disease are closely related.Ubiquitin-proteasome pathway includes ubiquitin,ubiquitin-conjugating enzyme(E2),ubiquitin-activating enzyme(E1),ubiquitin ligase (E3) and the 26S proteasome.Ubiquitin-proteasome pathway is very complex.A ubiquitin protein comprise76 amino acids which contas seven internal lysine residues(K6,K11,K27,K29,K33,K48 and K63).These lysine acid residues are the connection site of polyubiquitin chain extension. Ubiquitin’s molecular weight is 40～50kD.Before the degradation of the protein,they must be marked by polyubiquitin chain.A problem of proteasome degradation may lead to the accumulation of abnormal protein and would therefore lead to production of Lewy bodies in dopaminergic neurons.Immunohistochemistry results showed thatα-synuclein is the main ingredient contained in this inclusion.α-synuclein is widely distributed in healthy brain tissue.The soluble protein includes 140 amino acids,is mainly located in the presynaptic neuron terminal areas.Under physiological conditions,α-synuclein may be to maintain the normal function of synapses,and involve in the regulation of dopamine biosynthesis.When injury occurs,α-synuclein aggregation may be adopted to resist injury.With theα-synuclein aggregation,abnormal proteins can not be cleared in time.Instead,they have a toxic effect on cells and accelerate cell death.At present,PD is divided into drugs treatment,neuroprotective treatment,as well as gene therapy treatment and neurological surgery.To date symptomatic treatment of Parkinson’s disease is still the main means of treatment.It is difficult to prevent or slow disease progression.In addition, long-term symptomatic treatment will bring a lot of complications. Therefore,to further explore the pathogenesis and prevention and treatment of Parkinson’s disease remains serious problems.Neurological disorders in humans can be modeled in animals using standardized procedures that recreate specific pathogenic events and their behavioral outcomes.The development of animal models of PD is important to test new neuroprotective agents and strategies.Such animal models of PD have to mimic,at least partially,a Parkinson-like pathology and should reproduce specific features of the human disease.Over the years,a broad variety of experimental models of PD were developed and applied in diverse species.Most PD(approximately 90%) is sporadic(sPD).Familial Parkinson’s disease accounts for only 5%～10%of the total number. Moreover,recent research suggests that pathogenesis of sporadic PD may be related to mitochondrial complexⅠ.Therefore,Parkinson’s disease animal model with inhibition of mitochondrial complexⅠshould be the best model.Recently,numerous in vivo and in vitro studies demonstrated that the herbicide rotenone produces specific dopaminergic cell degeneration accompanied by the formation of alpha-synuclein fibrils,cytoplasmic inclusions similar to those found in PD patients.Alpha-synuclein is a constituant of Lewy bodies,the hallmark of idiopathic Parkinson’s disease. The destruction of dopaminergic neurons by rotenone is attributed to an inhibition of the neuronal mitochondrial complexⅠ.Rotenone is a natural pesticide in vegetables,fruit trees and medicinal crops.It is in a wide range of applications.Chronic exposure of rotenone can cause environmental pollution.Because rotenone is high lipophilic,it can go easily through the blood-brain barrier.Then it will act on brain mitochondrial respiratory chain complex(ComplexⅠ).The delivery of respiratory chain in cells will be blocked.Rotenone is different from other’s complexⅠinhibitor,it can play a role in its toxicity directly without the need of the dopamine transporter,administration of rotenone in rats also occur Parkinson’s disease-like catalepsy clinical Performance. Therefore,rotenone may be one of the potential pathogenesis of Parkinson’s disease.Well,as soon as possible to establish a stable Parkinson model of animal studies will be of great significance to understand the role of the environmental factors in pathogenesis of Parkinson’s disease In recent years, many researchers established rotenone rat models of Parkinson’s disease.Due to complex experimental conditions and high mortality,it is difficult to promote large-scale application,so the purpose of our experiment is to improve the rotenone rat model.Methods:Sprague-Dawley male rats(180-220 g) were provided.The animals were divided in the follow groups:(1)Gradient dose model group(n=40), animals were first infused with 2mg/kg rotenone for 2days,followed by rotenone 1mg/kg dosing four days,the last dose of 0.5mg/kg rotenone for 22 days.(2)Constant dose rotene group(n=20),animals were infused with 0.5mg/kg dose rotenone for 28 days.(3)Vehicle group(n=14),animals were infused with dimethyl sulfoxide(DMSO) for 28 days.(4)Saline control group(n=6):animals were infused with saline for 28 days.To observe the general state and to get body weight Once a week.Before killing the rats,behavioral experiments were enforced.Contents of dopa- mine and its metabolites in striatum were detected by HPLC method Tyrosine hydroxylase(TH) immunohistochemistry in substantia nigra was carried out.heart,liver,spleen,kidney were observed by HE staining.The morphological changes of hippocampus and parietal cortex were observed by.Nissl staining.Protein contents of ubiquitin andα-synuclein in the substantia nigra and striatum were detected by Western blot method.At the same time,TH/ubiquitin and TH/α-synuclein immunofluorescence double staining were exerted.Results:1.The rats in gradient dose rotenone group had poor general state. Body weight was reduced daily.Behavioral obstacles appeared.Results of HPLC test showed that the dopamine in striatum was decreased more than 80%.Dopamine metabolites also decline.Results of tyrosine hydroxylase immunostaining in substantia nigra also showed that the number of positive neurons and nerve fibers decreased significantly.Rats in constant dose model group had no behavioral changes.Contents of dopamine and its metabolites were in normal level,but injury appeared in the substantia nigra dopaminergic neurons.2.HE staining results showed that no difference could be seen in heart, liver,spleen,kidney.No changes happened in parietal cortex and hippocampi.The comman staining including tyrosine hydroxylase immunostaining and nissle staining in the substantia nigra results showed that nissl bodies were no difference in brain areas outside the substantia nigra part in different groups.3.Results ofα-synuclein/ubiquitin western-blot detection in substantia nigra and striatum showedα-synuclein/ubiquitin expression were increased in substantia nigra from two model groups.no difference in striatum could be appeared between groups.The results ofα-synuclein/ ubiquitinand tyrosine hydroxylase immunofluorescence double staining were same to western-blot detection.Conclusion:1.The subcutaneous injection method with low-dose rotenone successfully established Parkinson’s disease rat model. 2.To reduce the rotenone dose could significantly improve dopaminergic neurons in substantia nigra and animal behavior.3.Subcutaneous injection with low-dose rotenone could selectively induce dopamine neurons damages in substantia nigra.4.The total contents ofα-synuclein were increased in substantia nigra from two model groups.It showedα-synuclein aggregation could help to damage dopamine neurons.5.Expressions of ubiquitin and ubiquitin protein were increased in substantia nigra from two model groups.It showed ubiquitin-proteasome system dysfunction was related to dopaminergic neuron damage.更多还原