【作者】 王诚；

【导师】 包仕尧；

【作者基本信息】 苏州大学 ， 神经外科学， 2007， 博士

【摘要】 帕金森病(Parkinson`s disease PD)是一种常见的中枢神经系统变性疾病,其病理改变的最大特点是病变只侵犯中脑的多巴胺(DA)能神经元,导致纹状体内DA的减少从而引起运动迟缓,静止性震颤,肌强直等锥体外系症状。目前药物治疗和手术治疗可使患者的症状在一定时间内获得不同程度的好转,但无法阻止本病的自然进展。随着神经生物学,分子生物学及移植免疫学等相关学科的发展,脑移植和基因治疗成为理论上治疗帕金森病最好的方法,其中通过移植干细胞替代受损的细胞,重建神经功能区和传导通路是近年研究的重点。干细胞根据其来源可分为胚胎干细胞和成体干细胞。骨髓基质细胞(Bone Marrow Stromal Cells,BMSCs)是一种位于成年动物骨髓的成体干细胞。在体外,BMSCs在一定的环境和条件下可分化为神经元样细胞,表达多种神经元特异性标志物。在体内, BMSCs可以穿越血脑屏障,与局部的组织整合,迁移到脑组织中的不同位置分化为不同类型的神经细胞。除此之外,BMSCs还具有自我更新和高度增殖等干细胞的特点。而且具有材料容易获得,分离培养较为容易,为同种同体细胞来源,无抗原性等优点,是神经系统疾病细胞替代治疗理想的靶细胞。本研究应用骨髓基质细胞体外培养、分离和扩增,用超顺磁性氧化铁微粒(Superparamagnetic iron oxide,SPIO)(商品名:菲立磁Feridex)标记后,定向注入大鼠纹状体、颈动脉、尾静脉移植治疗帕金森病大鼠模型,应用磁共振对移植的细胞进行活体示踪,结合病理学,观察骨髓基质干细胞体内迁移、脑内存活和神经分化以及帕金森病大鼠症状的改善情况。第一部分大鼠骨髓基质细胞的体外培养及诱导分化的研究目的:探索大鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)的体外培养和诱导分化方法。方法:体外分离SD大鼠骨髓,贴壁法扩增培养大鼠BMSCs,相差显微镜观察其生长特性及形态变化,传代至第4代后用神经营养因子诱导分化,在诱导后第3天和第7天分别行神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)、酪氨酸羟化酶(TH)、胶质原纤维酸性蛋白(GFAP)免疫细胞化学进行鉴定。结果:培养的BMSCs在第4代纯度在95%以上,大鼠BMSCs经表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、脑源性神经营养因子(BDNF)、全反式维甲酸(TRA)等诱导后能表达nestin、NSE、GFAP等特异性标记物,未出现TH染色阳性细胞。结论: BMSCs在体外扩增迅速,易纯化,联合应用细胞因子可诱导BMSCs形成神经干细胞,并向神经元分化。第二部分应用6-羟基多巴胺建立帕金森病大鼠模型研究目的:建立简单、易行、成功率高的偏侧帕金森病大鼠模型。方法:SD大鼠80只,注射同等剂量的6-羟基多巴胺(6-OHDA)于内侧前脑束不同部位。位点1:前囟后1.8mm、右侧2mm、硬膜下8.5mm;位点2:前囟后1.8mm、右侧2mm、硬膜下7.5mm,制作黑质完全损伤型PD大鼠模型,运用阿朴吗啡诱发旋转试验及免疫组化检验模型是否成功。磁共振活体检测PD大鼠黑质、纹状体的毁损情况。结果:注射6-OHDA后第4周内有54只大鼠诱发明显的旋转行为,且旋转次数>7转/min,模型成功率为75%。MRI显示模型大鼠第1周后毁损侧黑质较对侧出现了明显的MRI低信号区,且随着时间的延长低信号区逐渐减小,至第3周已基本消失。结论:应用6-OHDA小剂量两点毁损内侧前脑束制备的偏侧帕金森病大鼠模型具有较高的成功率,是帕金森病研究较为理想的模型。MRI扫描可以活体连续观察帕金森病大鼠模型的毁损情况,是客观评价和检测帕金森病大鼠模型的一种有效工具。第三部分菲立磁标记骨髓基质细胞治疗帕金森病大鼠及磁共振示踪目的:探讨应用骨髓基质细胞治疗帕金森病大鼠的机制及疗效,以及菲立磁(Feridex)标记的BMSCs移植入PD大鼠体内后,磁共振示踪观察的可行性。方法:将45只帕金森病SD大鼠分为尾静脉移植组、颈动脉移植组和脑内移植组,移植Feridex和5-溴脱氧尿核苷(BrdU)双标记的BMSCs。另5只帕金森病SD大鼠为未治疗对照组。移植后2、4、6、8周进行阿朴吗啡诱发旋转试验和MRI示踪观察,成像后相应时间点每组处死2只大鼠,取脑组织冰冻切片后进行普鲁士蓝染色及免疫组化染色。结果:尾静脉移植组、颈动脉移植组和脑内移植组第4周阿朴吗啡诱发旋转试验较未治疗对照组有明显减少;其中,脑内移植组减少最明显,优于颈动脉移植组和尾静脉移植组。组织学和MRI示踪发现移植的BMSCs在帕金森大鼠脑内存活、迁移,免疫组化染色提示移植的BMSCs在脑内能向神经细胞方向分化。结论:应用BMSCs治疗帕金森病大鼠疗效显著,菲立磁可以用来体外标记骨髓基质细胞,利用MRI技术可以对脑内移植后的标记细胞进行初步的活体追踪。更多还原

【Abstract】 Parkinson’s disease (PD) is a progressive neurodegenerative disorder, which is primarily characterized by degeneration of the dopaminergic neurons of the nigrostriatal pathway. Although levodopa is still considered as the gold standard of antiparkinsonian drug therapy, chronic levodopa treatment is associated with the development of adverse events in the majority of patients, such as motor fluctuations, dyskinesias, and neuropsychiatric problems. Nowadays, most of the PD research has been focused on cell transplantation, which could be a potential therapeutics.Bone Marrow Stromal Cells (BMSCs) are stem cells with the characteristics of self-renewing, multi-potency and easily expanding in vitro. Therefore, BMSCs are ideal target cells for cell transplantation. Under specific conditions, BMSCs are capable to differentiate into other cell types, such as neural stem cells, neural cells and glial cells. A study has shown that there was a functional recovery with BMSCs transplantation in a PD animal model. BMSCs are able to differentiate into neural stem cells, neural cells and glial cells and dopamine cells when they are transplanted into caudate putamen of PD rats. Our research was focused on: (1) Cultivation,differentiation and identification of BMSCs from SD rats.(2) Establishing 6-HODA PD model with simple method. (3) Transplanting BMSCs labeled with Feridex and bromodeoxyuridine (BrdU) to treat Parkinson’s disease in SD rats. (4) To examine the effect of treatment with BMSCs in Parkinson’s disease rat model, and explore the methods of labeling bone marrow stromal cells with Feridex and to monitor the labeled cells after transplantation into the PD rats with MRI scanning.Part I Cultivation and Differentiation of BMSCs in VitroObjective:To explore the cultivation,differentiation and identification of BMSCs from SD rats.Methods:BMSCs were isolated from SD rats by anchoring culture.The proliferative characteristics were observed in primary and passage cultures. High purified BMSCs were selected and induced into neurons through the neurotrophic factors.The number of different immunoreactive cells was detected by nestin,NSE,GFAP and tyrosine hydroxylase (TH) immunocytochemistry.Results:The fourth generation cells were highly purified BMSCs. EGF, bFGF, BDNF and TRA might induce BMSCs into nestin,NSE and GFAP-positive cells, but no TH-positive cell was found. Conclusion:BMSCs can be amplified and purified in short time.Combination of cytokines may have synergic effects on proliferation and differentiation of BMSCs.PartⅡStudy on Injection 6-OHDA into Medial Fore Brain Bundles and Establish the Rat Model with Parkinson’s DiseaseObjective:To establish a simple and effective rat model in Parkinson`s disease. Methods:80 SD rats were selected. 6-OHDA was injected into two points of medial forebrain bundles on the right sides. One burr hole was made, two injection points are–1.8mm antero-posterio to and–2mm lateral to the bregma, -8.5mm ventral to the dura mater and–1.8mm antero-posterio to and–2mm lateral to the bregma, -7.5mm ventral to the dura mater. In 6 weeks, apomorphine-induced rotation test was performed to examine the disease progress in this rat model.Further examined the lesions of substantia nigra and striatum of PD rats in vivo under MRI. Results:54 rats were induced to show obviously rolling behavior at the fourth week after lesion of substantia nigra (>7 rolls/min).The Success ratio of PD model was 75%.The lesioned substantia nigra of PD rats showed obviously low MRI signal.The low signal regions shrank in correspondence with the time extended, and disappeared at the end of the third week. Conclusions:The PD rat model gotten from bi-point lesions of medial forebrain bundles by small dose of 6-OHDA is effective with a high success rate. It’s a perfect model to study PD.MRI scanning can be used to observe the lesions of PD rat model in vivo continually, which is an available means to evaluate and examine the PD rat model objectively. Part III Transplanted Bone Marrow Stromal Cells labeled with Feridex In Rat Parkinson’s Disease Models by MRI measurementObjective: To examine the effect of treatment with BMSCs in Parkinson’s disease rat model, and explore the methods of labeling bone marrow stromal cells with Feridex and to monitor the labeled cells after transplantation into the PD rats with MRI scanning. Methods: 45 PD rats were randomly divided into three groups: intravenous group、intra-arterial group and intracerebral group.They were transplanted with BMSCs labeled with Feridex and bromodeoxyuridine (BrdU). Another 5 PD rats were untreated group. Functional outcome measurements were performed using the apomorphine induced rotation test at 2,4,6 and 8 weeks after treatment. At the same time MRI scanning was performed to monitor the transplanted cells. After MR imaging,two rats of each group were killed and Prussian blue staining and immunofluorescent staining of the histological sections were performed. Results: A decrease of per minute numbers of apomorphine induced rotation test was noted in rats of intravenous group、intra-arterial group and intracerebral group.The decrease was more significant in intracerebral group. BMSCs colabeled with Feridex and BrdU could migrate into the lesions and differentiate after transplanted into PD rats. MRI scanning is a useful and noninvasive tool to track the location and distribution of the labeled BMSCs. Conclusions: Treatment with BMSCs may improve neurological outcome in PD rat model. Fefidex can be used to label BMSCs in vitro and MRI opens up the possibility of in vivo tracking of Fefidex-labeled BMSCs after transplantation.更多还原