【作者】 朱庆丰；

【导师】 袁崇刚；

【作者基本信息】 华东师范大学 ， 生理学， 2010， 博士

【摘要】 帕金森病（Parkinson’s disease,PD）是一种发生于中老年期的,缓慢进展的神经系统退行性疾病,主要病理改变为中脑黑质致密部（substantia nigrapars compacta,SNc）多巴胺（dopamine,DA）能神经元变性死亡,导致纹状体来自黑质的DA能神经末梢减少,从而引起震颤、肌肉僵直、运动迟缓与体位不稳等一系列综合病症（Lotharius and Brundin,2002）。PD是继阿尔茨海默病（Alzheimer’s disease,AD）之后第二大常见的神经系统退行性疾病,随着人口平均寿命延长和老龄化出现,PD的发病率呈现明显的上升势头。药物治疗以及脑深部电极（deep-brain stimulation,DBS）埋藏治疗可在一定程度上缓解患者的症状,但都存在一些副作用,而且不能从根本上抑制脑内DA能神经元的持续性死亡。近年来神经干细胞（neural stem cells,NSCs）的研究为细胞替代疗法治疗PD提供了潜在的应用前景,通过移植神经干细胞不但可以来替代部分丢失的的DA能神经元的功能,而且可以通过移植具有分泌神经营养因子特性的细胞抑制DA能神经元的丢失,长期有效地改善患者的症状。在本研究的第一部分,我们利用本实验室建立的胚胎大鼠NSCs的取材和培养方法,分离E14 SD大鼠胚胎前脑室下带区（subventricular zone,SVZ）神经组织,在含有多种神经营养因子的无血清培养基中悬浮培养,经传代后培养出大量NSCs标记分子——神经巢蛋白（neuro epithelial stem cell protein,Nestin）阳性克隆球团。经免疫组织化学方法检测,这些阳性细胞克隆球团经诱导分化后可以分化为神经元、星型胶质细胞和少突胶质细胞,此结果说明本实验取自E14 SD大鼠SVZ区域的细胞具有自我增殖和多潜能分化特性。为长期跟踪移植细胞在体内存活、生长、迁移及分化的过程,我们运用用携带有增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因的重组腺相关病毒（rAAV₂-EGFP）在体外培养条件下感染NSCs,通过病毒在细胞内表达EGFP使细胞带有绿色荧光标记。实验中发现,被AAV₂-EGFP感染后的NSCs仍具有持续的分裂增殖能力。EGFP标记的NSCs体外诱导分化后,整个细胞从胞体到突起末梢都带有绿色荧光标记,较好显示出了细胞的完整形态,经测定,体外EGFP标记率为65%。在第一部分研究,我们成功建立了NSCs的分离、培养、鉴定和EGFP标记示踪的方法。PD在动物中没有自发倾向,进行PD实验研究需要建立适当的动物模型（陈生弟,陈先文,2003）。建立稳定的能模拟人类PD病理学和行为学改变的动物模型,对PD的发病机制和治疗等方面的研究具有重要的意义。PD大鼠模型由于可直接观察行为学改变、经济和易于操作等原因,成为目前应用最广的研究模型。现在PD模型的制作方法较为成熟,常用的是6-羟基多巴胺（6-hydroxydopamine,6-OHDA）损毁模型。在第二部分实验中,我们采用了单点内侧前脑束（medialforebrain bundle,MFB）注射6-OHDA方法制备一侧黑质损毁PD动物模型。共制备模型105只SD大鼠（Sprague Dawley,SD）。建模4周后经阿朴吗啡（apomorphine,APO）诱发旋转,旋转7 r/min以上者为成功模型,共有57只,行为学检测造模成功率为54%。模型动物造模成功后分别在30,60和120天后再次进行行为学检测以跟踪动物的自我恢复可能性,统计结果显示与造模成功后的初次检测结果无显著性差异（P＞0.05）,表明PD模型稳定,没有自我修复现象。对PD模型组动物中脑黑质酪氨酸羟化酶（tyrosion hydrozylase,TH）免疫组织化学检测发现,成功PD大鼠中脑黑质正常侧TH反应阳性神经元数量较多,胞体较大,突起明显;而6-OHDA损毁侧TH阳性神经元数量则明显减少,细胞丢失超过90%以上。120天后PD模型动物损伤侧神经元数量减少仍然在90%以上,从组织学上证明模型的稳定。我们对成功的PD模型大鼠和正常大鼠分别在黑质或纹状体移植rAAV₂-EGFP标记的NSCs,移植后30、60和120天进行行为学检测。结果表明,NSCs移植到PD大鼠黑质或者纹状体,随着移植细胞在宿主脑内存活时间的延长,动物的行为学症状得到显著改善（P＜0.05,P＜0.01）。在NSCs移植疗法中,NSCs作为一种外源性物质能否在损伤的黑质区域存活或者迁移,能否向黑质特定DA能神经元分化是细胞移植治疗PD疗效的关键。在第三部分实验中我们研究了移植细胞的行为变化。在细胞移植后30、60和120天不同时期取脑进行连续切片,在移植后不同时间的脑片上我们均发现了大量EGFP标记的细胞,说明EGFP作为示踪记在细胞移植后仍然长期稳定表达,外源性的NSCs可以通过异体移植在宿主内长期存活。对各时间段有移植细胞标记的脑片进行免疫组化检测,结果显示移植的细胞可以分化为多种神经元,星形胶质细胞和少突胶质细胞。通过EGFP的示踪显示,移植细胞具有迁移现象并存在一定规律。移植到黑质的细胞随着时间的延长,在黑质区迁移的距离也越远,但总体上移植细胞是在受损的黑质区域内迁移,只有少量的细胞迁移到黑质区域以外。移植到纹状体的细胞呈条索状排列从移植位点向腹后内侧黑质部位进行较为广泛的长距离迁移,迁移具有明显的方向性并在黑质致密部可见散在移植细胞。实验结果提示移植的NSCs可以在体内存活并有迁移和分化能力,CNS损伤区微环境对移植细胞的迁移和分化具有一定诱导作用。综上所述,我们得出以下结论:1,从E14天胚胎大鼠SVZ分离出来的神经细胞群,能不断的更新和增殖,具有良好的可传代性,体外培养可以诱导分化为神经元,星形胶质细胞和少突胶质细胞等多种类型,具有自我增殖和多潜能分化的干细胞特性。利用rAAV₂-EGFP感染NSCs,可以使NSCs带有稳定绿色荧光标记,标记后NSCs仍具有增殖和分化特性。成功建立了胚胎大鼠NSCs的培养、鉴定和EGFP示踪标记方法。2,单点MFB注射6-OHDA制备一侧黑质损毁PD动物模型,应用免疫组化和行为学检测证明模型制作成功、可靠并可以长期保持稳定。用体外培养的rAAV₂-EGFP标记的NSCs移植PD模型大鼠纹状体或者黑质,随着移植细胞在宿主脑内存活时间的延长,可以明显改善PD动物行为学症状。3,跟踪NSCs移植后不同时间段移植细胞的分化和迁移特性。移植细胞在宿主脑内分化为多种神经元,星形胶质细胞和少突胶质细胞。移植细胞具有迁移现象并存在一定规律。移植到黑质的细胞总体上在受损的黑质区域内迁移;移植到纹状体的细胞呈条索状排列从移植位点向腹后内侧黑质部位进行较为广泛的长距离迁移,具有明显的方向性并在黑质致密部可见散在移植细胞。提示CNS损伤区微环境对移植细胞的迁移和分化具有一定诱导作用。更多还原

【Abstract】 Parkinson’s disease （PD） is a progressive neuro degenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta （SNc） and the consequent loss of projecting nerve fibers in the striatum produces the classical symptoms of resting tremor, bradykinesia, rigidity and postural instability etc. PD is one of the most common neuro degenerative disorders and has significantly the incidence of the upward trend with the extension of average life expectancy and population aging. Conventional pharmacological therapy and deep-brain stimulation （DBS） only temporarily reduce or alleviate the symptoms, but can not stop the process of dopaminergic neurons loss, long-term decline in efficacy, side effects increase. Neural stem cells （NSCs） provide attractive prospects for transplantation in neuro degenerative diseases. NSCs transplantation can not only complement the dopaminergic neurons loss, but also secrete neurotrophic factor to inhibit the loss process, long-term effective in improving symptoms of PD patients.At the first part of the study, we use the ways of our laboratory set up to separate and cultivate the embryonic 14 d （E14） rats NSCs which were obtained from the forebrain subventricular zone （SVZ）. The SVZ pieces were incubated in DMEM/F12 culture medium consisting of some neurotrophic factors without serum. The neurospheres were passaged weekly after dissociation and the cells were grown and expanded in flasks for several weeks. They demonstrated typical characteristics of stem cells including clonality, nestin-positivity and the ability to differentiate into any neural cell type in the CNS. The NSCs were labeled by enhanced green fluorescent protein （EGFP） using recombinant adeno-associated virus （rAAV₂） containing the marker gene of EGFP and still have a proliferation capacity after infection by AAV₂-EGFP. After differentiation, the EGFP labeled cells show a better morphological integrity from the cell body to neurite endings with a green fluorescent marker and in vitro the ratio of the cells with EGFP marker was about 65%. From this part, we successfully set up the method of isolation, culture, identification and marking tracer of NSCs.PD without spontaneous tendency in animals, it is required to establish the appropriate animal models to carry out experimental study. A stable animal model PD with analog of human PD pathology and behavioral changes is of great significance to the study of pathogenesis and treatment. PD rat becomes the most widely used experimental research model because of its economic, easy to operate and the easy testing of its behavior change etc. The methods of PD model production are now more sophisticated and commonly made by 6-hydroxydopamine （6-OHDA） administrtion. At the second part of our study, the adult Sprague-Dawley（SD） rats were used to establish PD models by unilateral microinjection of 6-OHDA which damaged dopaminergic neurons in the substantia nigra. Stereotaxic injections of 6-OHDA were performed in the right MFB of 105 adult SD rats. After 4 weeks, 57 rats were selected as successful PD models based on the presence of apomorphine-induced rotational behavior （>7 rpm）. The success rate for production of the 6-OHDA-induced PD model was 54%. The effects of the PD models’ stability were evaluated during the first, second, and fourth months. There was no improvement in rotational scores in any PD rats at any time point（P >0.05）. The extent of the 6-OHDA lesions in the MFB was verified by immunohistochemistry using an anti- tyrosion hydroxylase （TH） antibody. Brain sections from both （lesioned and unlesioned） sides of the SNc were compared simultaneously. The neurons are in larger quantities, larger cell body and obvious neurite in the non-lesioned area. A significant reduction more than 90% in TH-positivity compared with the non-lesioned area （left side） was seen in the lesioned area （right side）. This phenomenon was also observed four months later and proved the model’s success and stability. The NSCs labeled by EGFP were transplanted into the SNc or striatum of PD rats or normal rats. PD rats grafted with NSCs in the SNc or striatum showed significant reductions in rotational behavior compared with the pre-transplantation period with the grafed cells survival in the host （P <0.05, P<0.01）.With NSCs transplantation for the treatment of PD, the transplanted cells, whether or not to have the capacity of migration and anchoring at the injury site specifically or survival and differentiation to dopaminergic neurons in SN, are the key issues to the effect of the treatment of PD. At the third part of this study, we explored the behavior of cells after transplantation. The EGFP labeled cells are found in brain slices at any time point. EGFP tracer as recorded in the cells is long-term expression and exogenous NSCs can survive in host brain for long term. The immunohisto-chemistry results showed that grafted cells can differentiate into neurons, oligodendrocytes and glial cells in brain slices at any time point. There was regularity about the migration of grafted cells. The grafted cells in the SN migrate farther the distance with time lengthens, but the overall are at the SN region migration, only a small number of cells migrated to the region outside the SN. The cells transplanted into the striatum were cord-like arrangement from the transplant site to the ventral posterior medial SN part through a more extensive long-distance migration and there were some scattered grafted cells at SN. These results suggest that transplanted NSCs can survive well in vivo and possess the ability to migrate and differentiate, with a high degree of plasticity. The migration and differentiation patterns of transplanted NSCs may be partially regulated by the injured brain.In summary, we have reached the following conclusions:1. The cultured NSCs origin from SVZ of embryonic 14 d SD rats had the characteristics of proliferation and passage ability. They demonstrated typical characteristics of stem cells including clonality and the ability to differentiate into any neural cell type in the central nervous system （CNS） . The NSCs were successfully labeled by EGFP using rAAV₂ containing the marker gene of EGFP and still had a proliferation capacity after infection. Successfully established the methods of culture and identification of NSCs from SVZ of embryonic SD rats. 2. To establish PD models by unilateral microinjection of 6-OHDA at MFB which damaged dopaminergic neurons in the substantia nigra. The effectiveness of the 6-OHDA lesion was confirmed by apomorphine-induced rotational behavior and verified by immunohistochemistry. The results demonstrated that methods of making PD models were successful. The PD rats grafted with EGFP labeled NSCs in the SNc or striatum showed significant reductions in rotational behavior compared with the pre-transplantation period.3. To observe the differentiation and migration characteristics of grafted NSCs, grafed cells were traced and detected at various time periods after transplantation. The grafted NSCs had the ability to differentiate into any neural cell type in the host brain and there was regularity in migration. The grafted cells in SN migrated farther the distance with time lengthens, but the overall were at the SN region. The grafted cells in striatum migrated through a more extensive long-distance to SN and there were some scattered grafted cells at SN. The results suggested that the lesion microenvironment in the adult brain may be one of the factors which improve the survival, differentiation and migration of grafted cells in CNS.更多还原