【作者】 冯蕾；

【导师】 刘树伟；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 帕金森病（Parkinson’s disease,PD）是一种好发于中老年人并严重影响其生活质量的神经退行性疾病,该病的主要病理变化是黑质多巴胺能神经元的进行性退变和缺失,致使纹状体内多巴胺水平异常降低。迄今为止,PD的病因和发病机制还未完全明确。研究表明,PD与遗传、环境、细胞凋亡、线粒体功能异常等有关。随着老龄社会的来临,我国的PD发病率也在逐年升高,严重威胁着老年人的生活质量和生命安全。目前对该病的预防和治疗虽然有许多方案,但效果都不够理想。近年来,脑内移植技术与转基因技术相结合治疗PD,成为一新的研究热点。嗅鞘细胞（olfactory ensheathing cells,OECs）是目前所发现的极少数中枢神经系统可以再生的细胞之一。OECs兼有雪旺氏细胞与星形胶质细胞的双重功能特点,它能伴随轴突从外周神经系统进入中枢神经系统,能在损伤段为再生神经纤维形成髓鞘,保护其不受局部抑制因子的影响;尚可分泌多种神经营养因子（如NGF、BDNF、NT-4、NT-5等）。OECs是当今国际上普遍认为用作细胞移植治疗中枢神经系统损伤与再生修复很有临床应用前景的候选细胞。但是神经损伤或病变后影响神经再生修复的因素极其复杂,OECs本身表达神经营养因子的水平较低,单纯依靠OECs移植难以满足治疗的需要。胶质源性神经营养因子（glial cell line-derived neurotrophic factor,GDNF）是目前发现的在体外支持运动神经元生长最有效的神经营养因子。不仅能在体外有效地促进多巴胺能神经元存活和分化,而且在体内,对黑质纹状体多巴胺能系统也有保护和修复作用,是目前防治神经元退化性疾病的最有前途的一种神经营养因子。但由于不能通过血脑屏障,为临床应用带来极大困难。本课题用6—羟基多巴（6-OHDA）损伤制作的PD体内和体外模型,观察了GDNF基因修饰的OECs移植对PD大鼠模型的疗效,并利用免疫荧光、RT-PCR、流式细胞分析和Western blot等方法对其机理进行了初步研究。旨在为GDNF基因修饰的OECs移植治疗PD提供实验依据,为PD治疗提供新思路。本实验分为4部分,摘要如下:第一部分嗅鞘细胞的培养、纯化和鉴定和生物学特征目的:探讨新生大鼠嗅球OECs的分离培养和纯化方法,并观察其形态学变化;观察不同浓度OECs培养上清（conditional medium,CM）对6-OHDA引起的PC12细胞凋亡的抑制作用,探讨其机制。方法:采用原代培养技术,从新生大鼠的嗅球分离培养OECs,用差速贴壁和阿糖胞苷抑制相结合方法纯化培养OECs,并用牛垂体提取液（BovinePituitary Extract,BPE）扩增OECs,倒置相差显微镜下观察OECs形态变化特点及生长情况,采用NGFRp75抗体行免疫细胞化学染色鉴定,根据NGFRp75免疫细胞化学染色统计培养的OECs的纯度。收集OECs培养上清,将不同浓度（25%、50%和75%）的OECs培养上清与100μM 6-OHDA共孵育PC12细胞,用MTT法测各组细胞活力;倒置显微镜观察细胞形态变化:Hoechst33342/PI双染观察细胞凋亡的形态学改变;流式细胞技术检测线粒体膜电位的改变;RT-PCR检测各组细胞bcl-2/bax mRNA表达差异。结果:成功培养出高纯度的OECs,体外培养的新生大鼠OB-OECs主要为双极或三极细胞,其突起细长。未经纯化则成纤维细胞生长迅速而占据优势。CM（25%、50%和75%）处理组较6-OHDA组相比,细胞活性增强,Hoechst33342/PI双染显示凋亡细胞的比例减少,可稳定线粒体膜电位,bcl-2/bax mRNA比值升高,两者有统计学差异,p＜0.05。结论:差速贴壁和Ara-C相结合的纯化方法是一种高效率、方便、经济的纯化培养OECs的方法。OECs培养上清对6-OHDA诱导PC12细胞有保护作用,其机理可能是OECs分泌的营养因子,抑制bcl-2/bax mRNA比值降低,稳定线粒体膜电位而发挥细胞保护作用。第二部分GDNF基因修饰的嗅鞘细胞的构建和生物学鉴定目的:构建pIRES2-EGFP-GDNF载体,建立OEC s-GDNF基因工程细胞。方法:用TRIZOL Reagent法从新生大鼠脑组织提取总RNA,用RT-PCR反转录出GDNFcDNA片段,回收纯化,将其与经SacⅠ和SmaⅠ双酶切的pIRES2-EGFP连接,应用分子克隆技术构建载体pIRES2-EGFP-GDNF。将此重组载体用脂质体Lipofectamine^TM2000转入OECs细胞,用G418筛选阳性克隆,构建出OECs-GDNF工程细胞。用RT-PCR、ELISA检测OECs-GDNF工程细胞GDNF的表达变化。结果:经测序检验,成功构建出pIRES2-EGFP-GDNF,GDNF基因无突变;筛选出OECs-GDNF工程细胞,RT-PCR检测显示OECs-GDNF工程细胞GDNFmRNA表达明显增高,ELISA检测OECs-GDNF工程细胞培养上清中GDNF明显增高,分泌量为15.2ng/10⁶细胞/24hr。结论:成功构建了pIRES2-EGFP-GDNF表达载体,经测序分析GDNF无突变。Lipofectamine^TM2000转染OECs方法简单,转染效率较高,经RT-PCR、ELISA分析OECs-GDNF工程细胞可高表达GDNF。第三部分GDNF基因修饰嗅鞘细胞移植对帕金森病大鼠模型的治疗作用目的:研究GDNF基因修饰的OECs对PD大鼠模型的治疗作用。方法:大鼠脑立体定位仪下,用6-OHDA两点注射右侧内侧前脑束制作单侧PD大鼠模型,注射等剂量的生理盐水的作为假手术组。将实验分为4组,每组6只:A假手术组;BPD模型组（纹状体注入等量生理盐水）;C OECs-GDNF组（纹状体内注入OECs-GDNF工程细胞）;D OECs组（纹状体内注入OECs）。术后2w、4w、6w和8w,分别观察阿朴吗啡诱导的行为学改变:术后8w用TH免疫荧光染色检测各组大鼠黑质部位TH阳性神经元数量及纹状体TH阳性纤维密度的改变,分析对PD大鼠模型的治疗作用。结果:OECs-GDNF工程细胞植入纹状体后,PD大鼠的旋转行为有所改善,细胞移植后4w、6w和8w,旋转圈数较PD模型组明显减少。荧光显微镜下显示移植细胞成活,标记细胞向周围分散,提示细胞移植后可向周围迁移。TH免疫荧光染色结果显示:PD模型毁损侧黑质部位TH阳性细胞明显减少,纹状体部位TH阳性纤维终末密度值明显降低;与模型组相比,OECs-GDNF组大鼠毁损侧黑质多巴胺神经元数量明显增多,纹状体部位TH阳性纤维的相对密度值明显增强,两者有统计学差异,p＜0.05;OECs组和PD模型组比较,无统计学差异,p＞0.05。结论:OECs-GDNF工程细胞对帕金森病模型大鼠有一定的治疗作用:能改善PD模型大鼠的旋转行为,对黑质、纹状体部位多巴胺神经元有一定的保护作用。第四部分GDNF基因修饰嗅鞘细胞移植对帕金森病大鼠模型的Bcl-2/Bax表达影响目的:研究GDNF基因修饰OECs移植对PD大鼠模型Bcl-2/Bax蛋白表达变化的影响。方法:实验分组同第三部分。细胞移植后8w,低温快速取出大鼠中脑腹侧组织,用Western blot方法检测各组大鼠中脑腹侧组织中Bcl-2/Bax蛋白表达的变化。结果:与假手术组比较,6-OHDA引起了PD模型组大鼠中脑腹侧组织Bcl-2蛋白表达降低,Bax蛋白升高,p＜0.01。与PD模型组比较,OECs-GDNF组中脑腹侧组织Bcl-2蛋白表达增高,Bax蛋白表达降低,Bcl-2/Bax蛋白比值升高,两者有统计学差异,p＜0.05。与PD模型组比较,单纯OECs组Bcl-2蛋白表达增高,Bax蛋白表达无变化,但无统计学意义,p＞0.05。结论:6-OHDA引起了PD模型大鼠中脑腹侧组织Bcl-2表达降低,Bax蛋白升高;OECs-GDNF工程细胞对PD大鼠的治疗作用可能与其抑制Bcl-2蛋白表达的降低、Bax蛋白表达升高,升高Bcl-2/Bax比值有关。结论和意义:本课题研究发现,OECs培养上清对6-OHDA诱导的PC12细胞有较强的保护作用,其作用机理可能是OECs分泌的细胞因子提高了细胞bcl-2 mRNA的表达,降低bax mRNA的表达,稳定线粒体膜电位,抑制了细胞凋亡;OECs-GDNF工程细胞移植对PD大鼠模型有治疗作用,可改善其旋转行为,其作用机理可能与抑制中脑腹侧组织内Bcl-2蛋白表达的升高、Bax蛋白表达降低,升高Bcl-2/Bax比值有关。本课题利用细胞培养、分子克隆、免疫荧光、RT-PCR和Western blot等实验技术,研究了OECs细胞对PC12的保护作用和OECs-GDNF工程细胞对PD大鼠模型的治疗作用,及其机制,在细胞和分子水平上为OECs-GDNF工程细胞治疗PD提供了实验依据。更多还原

【Abstract】 Parkinson’s disease（PD）is a widespread neurodegenerative disorder among the old people,which seriously affects the health of individuals.Pathologically,PD experiences a progressive loss of dopaminergic neurons within the substantia nigra pars compacta and a reduction of dopamine in the striatum.So far,its etiology and etiopathogenesis is still unknown.Researchers proposed that heredity,environment, apoptosis and mitochondrial dysfunction were probably of the main mechanisms. Because of the aged tendency of population,the incidence of PD is growing year by year,threating the quality of life for the elder and forming an increasing economic burden for the society.There is no effective treatment up to date.Cell transplantation is a new therapeutic strategy for PD.However,the therapeutic effect of long time is inconsistent and some immune response often occurred in host brain. Recently,some researchers attempted to combine cells transplantation with gene transfer as a therapeutic application for PD.Olfactory ensheathing cells（OECs）are special population of glial cells sharing the properties with both Schwann cells（SCs）in peripheral nervous system（PNS） and astrocytes in central nervous system（CNS）,in which they accompany and surround the olfactory sensory axons from their peripheral origin in the olfactory mucosa across the cribriform plate and into the olfactory nerve fiber layer of the olfactory bulb.On one hand,OECs can protect the regeneration of axons from the influence of inhibitors and the remeylinization for axons;on the other hand,OECs can synthesise several trophic factors,such as nerve growth factor（NGF）,brain derived neurotrophic factor（BDNF）,neurotrophin 4/5（NT-4,NT-5）et al.More and more people are interested in these characters of OECs and look on them as the main transplantable cells.So OECs may be the better candidates for biological implantation,but the number of regenerating axons distal from the injury site was still limited partly because of the limited expression of neurotrophic factors and the differences in the capacity of various axonal populations to regenerate through OECs implants.Glial cell line-derived neurotrophic factor（GDNF）,a member of transforming growth factor（TGF-β）super-family,which was extracted and purified from B49 cells of adult rats by Lin in 1993,is a new kind of neurotrophic factor.GDNF is the most potent motor neurotrophic factor among neurotrophic factors found so far. GDNF has been reported not only to support the survival and differentiation of dopaminergic neuron,but also to protect and recover the dopaminergic system of substantia nigra and striatum in vivo.Nevertheless,there is a critical restriction on clinical application for GDNF because it can’t pass the blood brain barrier（BBB）.In this study,the models of PD made by 6-hydroxybutyrate（6-OHDA）in vivo were widely utilized.We adopted genetic technology and cell transplantation to treat PD,which is advanced in the world.At first,OECs were primarily cultured and purified in vitro.Then OECs-GDNF was built and transplanted into PD model rats. At last we investigated the mechanism of neurons protection of OECs-GDNF.Part 1 Culture,purification and biological characteristics study of OECsObjective:To investigate an effective method of purification for establishing primary cultures of OECs from the new born rat olfactory bulb and investigate the morphological changes and biological characteristics of the cultured OECs.Methods:OECs were harvested from olfactory bulbs and cultured,purified by the method of combining both the different rates of attachment among the various cells types and the Ara-c inhibition.The morphological changes of cultured OECs were observed at diferent stages and identified by nerve growth factor receptor p75 （NGFRp75）immunohistochemistry staining.We examined the effects of newborn rat OECs CM on PC12 cells treated by 100μM 6-OHDA.Cells were determined by MTT assay,Hoechst 33342/PI staining,Rh 123 staining and the ratio of bcl-2/bax mRNA expression by RT-PCR.Results:The cultured OECs presented two morphological types:fusiform, process-bearing multipolar and their processes weaved into net.OECs CM（25%, 50%,75%）was able to reduce the cellular damage in PC12 cells.CM could inhibit the disruption of mitochondrial transmembrane potential,upregulation of bcl-2 and downregulation of bax,compared with 6-OHDA alone,p＜0.05.Conclusions:The method of purification for OECs through combining both the different rates of attachment among the various cells types and the Ara-c inhibition is simple,inexpensive and practical.CM has a neuroprotective effect on 6-OHDA induced apoptosis of PC12 cells through up-regulation of the Bcl-2/Bax ratio and protection for mitochondrion.Part 2 Building engineered OECs-GDNF and its biological assayObjective:To construct recombination eukaryotic expression vector pIRES2-EGFP-GDNF and build the engineered OECs-GDNF.Methods:GDNF gene encoding fragment was amplified using RT-PCR and was cloned into the eukaryotic expression vector pIRES2-EGFP.After PCR amplification,SmaⅠ,SacⅠdigestion and DNA sequencing confirmation,the recombination eukaryotic expression vector pIRES2-EGFP-GDNF was constructed successfully.Then,we transfected GDNF into OECs with Lipofectamine^TM2000 positive-ion liposome,and built OECs-GDNF cells by using G418 culture medium to select positive clones.Finally,we assayed GDNF activation and quantity by ELISA and RT-PCR.So the OECs secreting high quantity of GDNF were genetic engineered cells.Results:PCR and DNA sequencing has verfied that the insert sequence was successfully cloned into the vector.ELISA assay demonstrated high GDNF protein secretion in OECs-GDNF conditioned medium.RT-PCR showed OECs-GDNF could up-regulate GDNF mRNA.Conclusions:Lipofectamine^TM2000 transfected pIRES2-EGFP-GDNF into OECs is convenient and has high transfection efficiency.Part 3 Therapeutic effect of OECs-GDNF transplanted into rat model of PDObjective:To study the therapeuti effect of OECs-GDNF transplanted into rat model of PD.Methods:After injecting 6-OHDA into the right medial forebrain bundle of rat for 2 weeks,we obtained the hemi-lateral model of PD.All rats were induced the rotational behavior after injecting apomorphine subcutaneously.The control group was set up by injecting isodose physiological saline as pseudo-operation rats.All the rats were divided into 4 groups:pseudo-operation group,PD model group, OECs-GDNF transplanted group and OECs transplanted group.The rotational behavior was evaluated by recording the numer of apomorphine-induced turns.We used tyrosine hydroxylase（TH）immunofluorescence staining to investigate the numbers of dopaminergic neurons in substantia nigra compacta（SNc）and the calibrated optic density value of TH positive fiber in striatum.Results:OECs-GDNF transplantation could ameliorate the rotational behavior of PD model rats,comparing with PD model group,p＜0.05.TH positive neurons in right SNc and TH positive fibers in right striatum of the PD model group significantly decreased,p＜0.01.After the transplantation OECs-GDNF,they could partly be restored,p＜0.05.Conclusions:OECs-GDNF transplantation had partial therapeutic effect on model rats of PD. Part 4 Effect of OECs-GDNF transplanted into PD rat on expression of Bcl-2/Bax proteinObjective:To observe the influence of OECs-GDNF transplantation on expression of Bcl-2/Bax protein in vivo.Methods:The experimental rats were divided into 4 groups according to part 3. In deeply anesthetized condition,ventral mesencephalic（VM）tissue were rapidly taken out on the ice,the Bcl-2/Bax protein expression was determined by Western blot.Results:Western blot showed protein expression of Bcl-2 in VM tissue of PD model rat decreased,p＜0.05.But it increased in OECs-GDNF transplantation group compared with PD model group at 8w.At the same time,protein expression of Bax increased in PD model rat and decreased in OECs-GDNF group.Bcl-2/Bax in OECs-GDNF group obviously increased compared with PD model group,p＜0.05.Conclusions:OECs-GDNF transplantation can up-regulate Bcl-2 protein and down-regulate Bax protein.OECs-GDNF transplantation could inhibit the depression of Bcl-2/Bax ratio to prevent the dopaminergic neurons from apoptosis.Conclusions and significance:In this study,OECs conditioned medium could afford significant neuroprotection against 6-OHDA-induced injury in PC12 cells via upregulation of bcl-2,downregulation of bax and thus attenuated the mitochondrial transmembrane potential loss,so the apoptosis was inhibited and the viability of PC12 cells was increased.OECs-GDNF transplantation had partial therapeutic effect on model rats of PD,which could up-regulate Bcl-2 protein and down-regulate Bax protein in PD model,inhibit the depression of Bcl-2/Bax ratio to prevent the dopaminergic neurons from apoptosis.Using the technology of cell culture,molecular clone,immunofluorescence staining,RT-PCR and Western blot,we research the neuroprotection on PC12 cells of OECs CM and the therapic effect on PD of OECs-GDNF transplantation.On cellular and molecular level,our study provided some experimental evidence on treating PD using OECs-GDNF.更多还原