【作者】 邓兴力；

【导师】 冯忠堂； 刘如恩； 杨智勇； 王廷华； 李力燕； 董坚；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 目的帕金森病(Parkinson’s disease,PD)为常见于中老年的神经退行性疾病,其主要病理改变为黑质致密部(substantia nigra pars compacta,SNpc)多巴胺(dopamine,DA)神经元的变性及其导致纹状体(striatum,Str)内神经递质DA的耗竭,目前尚无可取得满意疗效的治疗手段。随着近年细胞生物学研究的不断深入,神经细胞移植技术日益受到人们的关注。由于PD病变区域较为局限,被认为是神经细胞移植治疗最有希望治愈的中枢神经系统疾患之一。尽管已有研究证实移植DA神经元可改善PD动物的运动症状,然而由于机体局部微环境的影响,导致移植DA神经元存活率较低,使其难以取得稳定的疗效。神经干细胞(neural stem cells,NSCs)作为神经组织特异性的前体细胞,具有来源丰富,增殖、分化能力强,能整合于宿主细胞,极低或无免疫源性,无致瘤性等特点,为PD的细胞移植治疗的带来新的希望。最近研究表明,源于胚胎中枢神经系统各区域的NSCs具有不同的分化潜能,中脑来源的NSCs(mesencephalic neural stem cells,mNSCs)更易于分化为DA神经元,是细胞移植治疗PD的理想细胞源。然而由于机体局部微环境的影响,仅少量mNSCs可在体内分化为DA神经元。胶质细胞源性神经营养因子(glial cell line derived neurotrophic factor,GDNF)是目前发现最强的DA神经营养因子,然而由于血脑屏障和脑组织结构的特点,使其应用受到了限制。随着现代分子细胞生物学技术的发展,基因治疗为解决GDNF给药途径问题带来新的希望。综上所述,为取得理想的疗效,PD的细胞移植治疗不仅需要替代局部缺失的DA神经元,更需要改善机体局部微环境,使其有利于移植细胞的存活、生长并向DA神经元分化。因此,本研究拟将DA神经元、mNSCs的移植替代作用和GDNF的DA神经营养作用相结合,探讨GDNF基因修饰大鼠胚胎mNSCs联合DA神经元移植对PD模型大鼠的治疗作用,为PD的细胞移植治疗提供新的思路。方法1.从U-251细胞总RNA中扩增GDNF编码序列,将其克隆至真核表达载体pEGFPN1构建重组质粒pEGFPN1-GDNF,经酶切鉴定及序列分析后,以FuGeneHD转染试剂介导转染COS-7细胞,应用RT-PCR、western blot和免疫细胞化学鉴定GDNF在细胞内的表达。2.解剖分离E14d大鼠胚胎腹侧中脑组织,经机械吹打制成单细胞悬液,接种于无血清培养基中培养,观察其增殖、分化并进行Nestin免疫细胞化学鉴定和诱导分化后β-Ⅲ-tubulin、GFAP、CNPase免疫细胞化学鉴定。3.采用核转染技术将重组质粒pEGFPN1-GDNF转入大鼠胚胎mNSCs,经G418筛选建立GDNF基因修饰大鼠胚胎mNSCs;以FuGene HD转染试剂介导超顺磁氧化铁(superparamagnetic iron oxide,SPIO)标记GDNF基因修饰mNSCs;应用RT-PCR、western blot和免疫细胞化学鉴定GDNF在GDNF基因修饰mNSCs中的表达;普鲁士蓝染色、透射电镜鉴定SPIO标记;体外诱导分化后免疫细胞化学鉴定其分化能力。4.分离培养大鼠胚胎中脑DA神经元:应用RT-PCR鉴定DA神经元标志基因的表达;β-Ⅲ-tubulin、TH免疫细胞化学鉴定的其纯度。5.中脑背盖腹侧区(ventral tegmental area,VTA)、内侧前脑束立(medial forbrainbundle,MFB)体定向注射6-OHDA建立PD模型大鼠。将PD模型大鼠随机分为DA神经元移植组、GFP基因修饰mNSCs移植组、GDNF基因修饰mNSCs移植组、GFP基因修饰mNSCs联合DA神经元移植组、GDNF基因修饰mNSCs联合DA神经元移植组和对照组;利用立体定向技术,将相应细胞移植到PD大鼠纹状体区。通过阿朴吗啡(APO)诱导PD大鼠旋转行为评估细胞移植的治疗作用。6.应用磁共振成像观察移植SPIO标记mNSCs在PD大鼠纹状体内的存活和迁移情况;免疫荧光组织化学研究移植mNSCs在PD大鼠纹状体内的存活、迁移和分化。结果1.重组质粒pEGFPN1-GDNF经双酶切产生650 bp和4700 bp的片段,测序分析结果证实DNA插入片段与文献报道完全一致。重组质粒pEGFPN1-GDNF转染COS-7细胞后,RT-PCR、western blot和免疫细胞化学结果证实GDNF能在COS-7细胞中正确表达。2.大鼠胚胎mNSCs原代培养7 d后,可形成大量悬浮生长巢蛋白(nestin)免疫阳性的神经球,经诱导分化后细胞呈微管蛋白β-3(β-Ⅲ-tubulin)、胶质纤维酸性蛋白(glial fibrillary acidicprotein,GFAP)或2’,3’-环腺苷酸-3’-磷酸二酯酶(2’.3’-cyclic nucleotide 3’-phosphodiesterase,CNPase)免疫阳性。3.重组质粒pEGFPN1-GDNF转染第三代大鼠胚胎mNSCs后,RT-PCR、westernblot和免疫细胞化学证实GDNF能在mNSCs中正确表达;普鲁士蓝染色、透射电镜证实SPIO成功标记GDNF基因修饰mNSCs;体外诱导分化研究表明GDNF基因修饰及SPIO标记不影响mNSCs的增殖和分化。4.大鼠胚胎DA神经元于体外培养7-11d细胞生长最为旺盛;RT-PCR结果显示细胞表达Nurrl、lmxlb和TH等DA神经元标志基因;免疫细胞化学表明分离培养的DA神经元纯度达85.7%。5.APO诱导大鼠旋转行为学评估证实成功建立偏侧PD大鼠模型,成模率达55.7%。细胞移植后APO诱导大鼠旋转行为学评估显示:与对照组相比,细胞移植能显著改善APO诱导PD大鼠的异常旋转行为(P＜0.01),以GDNF基因修饰mNSCs联合DA神经元移植疗效最为明显,细胞移植后旋转圈数下降到移植前的24%(P＜0.01)。6.磁共振T2 FFE扫描图像显示SPIO标记细胞移植区呈低信号改变,随时间延长可见低信号区向周围扩大。普鲁士蓝染色结果显示,各组PD大鼠纹状体内可见大量SPIO标记细胞停留于移植原位,仅有少数细胞向周围脑组织迁移。8.免疫荧光组织化学染色结果显示:各细胞移植组纹状体内可见大量细胞停留于移植原位,仅有少数细胞向周围脑组织迁移;移植后大多数mNSCs仍保持其未分化状态或分化为神经胶质细胞,仅少数细胞可分化为DA神经元;与其它各组相比,GDNF基因修饰mNSCs联合DA神经元移植组有更多的mNSCs分化为DA神经元。结论1.成功建立了GDNF真核表达质粒pEGFPN1-GDNF和GDNF基因修饰mNSCs。2.SPIO可用于体外标记mNSCs,通过MRI成像可对脑内移植的SPIO标记细胞进行初步活体示踪。3.与mNSCs或DA神经元单纯移植相比,GDNF基因修饰mNSCs联合DA神经元移植可显著改善PD大鼠的异常行为,但其作用机制有待进一步研究。更多还原

【Abstract】 OBJECTIVEParkinson disease（PD）is a chronic neurodegenerative disorder characterized by tremor,rigidity,and hypokinesia.The main pathology underlying disease symptoms in PD is a rather selective degeneration of nigrostriatal neurons leading to severe loss of dopamine（DA）in the stritum.At present,there’s no therapy method which has satisfactory curative effect on PD.As the development of cell biology,the cell replacement stratage becomes more and more attractive.PD,with pathology changes with limited area,seems to be one of the diseases which can be cured by cell transplantation.Numerous researches show transplantation of DA neurons can improve the abnormal behavior of PD animals. However,the therapeutic effect of DA neuron transplantation in PD is not stabilize because of the low survival rate of transplanted DA neurons induced by local microenvironment of host.Neural stem cells（NSCs）are a subtype of tissue-specific progenitor cells that is capable of extended self-renewal and the ability to generate all major cell types of nervous tissue,such as neurons,astrocytes and oligodendrocytes. NSCs are considered as one of the most promising cell sources in cell therapy of central nervous systerm（CNS）diseases because of their unique superiorities including abundant resources,strong ability of proliferation,can integrate with host cells,without immunogenicity and oncogenicity,etc.Recent studies show that NSCs derived from different region of embryonic CNS have different potential of differerntiation.Compare with other NSCs,midbrain derived NSCs（mNSCs）have more tendancy to differentiate into DA neurons.Therefore,it is thought that mNSCs is the reasonable cell source for cell therapy in PD.However,only few mNSes can differentiate into DA neurons in vivo due to the effect of local microenvironment of host.Glia cell line derived neurotrophic factor（GDNF）is the most powerful trophic factor for DA neurons.However,its application is limited because of difficulty to cross blood-brain barrier.As the development of molecular biotechnology,gene therapy,as the most promising strategy to solve the problems in administrationg route of GDNF, become a new direction for therapy research of PD.In summary,in order to obtain reasonable therapeutic effect,the target of cell therapy for PD is not only replace local missing DA neurons,but more important,is to improve the local microenvironment of host,made which be fit for survival and growth of transtrantated cells and differentiated into DA neurons.Therefore,this study will explore the therapeuric effect of intracerebral transplantation of GDNF gene modified mNSCs combination with DA neurons in 6-hydroxydopamine （6-OHDA）rat model of PD.METHODS1.The coding sequence（CDS）of GDNF was emplified by RT-PCR from human astrocytoma cell line U251.By gene recombination technique,GDNF CDS was inserted into eukaryotic expression vector pEGFPN1 to construct recombinant plasmid pEGFPN1-GDNF.The recombinant piasmid was identified with restriction enzyme digestion and DNA sequencing.COS-7 cells were transfected with the recombinant plasmid by Fugene HD transfection regent.The expression of GDNF was analyzed by RT-PCR,western blot as well as immunocytochemistery.2.The ventral mesencephalon was dissected from embryonic day 14（El4）rat embryo. By mechanical separation method,the brain tissue was triturated into a fine single-cell suspension.The cells were cultured with the serum-free medium containing DMEM/F12（1:1）,N2 supplement,EGF and bFGF.After primary neurospheres formed,cells were sub-cultured.The neural spheres of the third passage were identified with immunocytochemistery for Nestin.At the same time,the cells were inducted to differentiation,and the phenol types of differentiated cells were identified by immunocytochemistery forβ-Ⅲ-tubulin,GFAP and CNPase respectively.3.The mNSCs of the third passage were transfected with plasmid pEGFPN1-GDNF using the nucleofaction technique,48 hoUrs after transfection,the transfected cells were screened with medium containing G418,the positive clones were selected and proliferated and then labeled with SPIO mediated by FuGENE HD transfection reagent.The expression of GDNF was analyzed by RT-PCR,western blot as well as immunocytochemistry.Prussian blue stain and transmission electron microscopy was used to identify the SPIO particles in cells.To identify the differentiation ability of SPIO labled GDNF gene modified mNSCs,immunocytochemistry forβ-Ⅲ-tubulin, tyrosine hydroxylase and GFAP were performed after in vitro differentiation.4.The ventral mesencephalon was dissected from embryonic day 14 rat embryo.By trypsin digestion and mechanical separation,the brain tissue was triturated into a fine single-cell suspension.The cells were cultured with the medium containing Neurobasal,1%fetal bovine serum and 2%B27 supplement.The expression of DA neuron-specific genes was identified by RT-PCR.The purity of cultured cells was detected by TH immunocytochemistry assay.5.The rat models of PD were established by unilateral stereotaxic injection of 6-OHDA into rat ventral tegmental area（VTA）and medial forebrain bundle（MFB） region.For cell transplantation,the PD rats were randomly devided into DA neurons transplantation group（n=6）,GFP gene modified mNSCs transplantation group（n=6）, GDNF gene modified mNSCs transplantation group（n=6）,GFP gene modified mNSCs and DA neurons cotransplantation group（n=6）as well as GDNF gene modified mNSCs and DA neurons cotransplantation group（n=6）.Respective cells were stereotaxic injected into striatum（Str）of PD rats.Apomorphine（APO）induced rotational behavior test was performed to evaluate the therapeutic effect of cell transplantation.6.MRI scan was carried out to tracking the survival and migration of transplanted SPIO fabled mNSCs in vivo,and immunofluorescence histochemistry was conducted to identify the distribution and differentiation of transplantated cells.RESULTS1.The RT-PCR product of GDNF coding sequence was 650bp specific segment.By restriction enzyme digestion,the recombinant plasmid vector was digested into 650bp and 4700bp fragments.The DNA sequence of the 650bp fragment was identical with human GDNF CDS in GenBank.The results of RT-PCR,western blot and immunocytochemistery showed the GDNF was expressed successfully in COS-7 cells.2.7 days after primary culture,the cells derived from E14 rat embryonic mesencephalon form neurospheres which can be sub-cultured.A great many of neurospheres can obtained by successive passage.Immunocytochemistery showed the neurospheres were nestin positive,after differentiation the cells expressedβ-Ⅲ-tubulin,GFAP,and CNPase.3.The expression of EGFP was initially found 12 hours after transfection,increased remarkable 24 hours after transfection and reached a summit at 48 hours.One month after screened with medium containing G418,the positive clones were formed. RT-PCR,western blot and immunocytochemistery showed the GDNF was expressed correctly in cells.Prussian blue stain showed numerous blue stained particles in the cytoplasma of the labeled cells.Transmission electron microscopy showed vacuolar structures of different sizes under the cytoplasma within and outside of which there were highly density particles.The immunocytochemistery showed the labeled cells were nestin positive,after differentiation the cells expressedβ-Ⅲ-tubulin,TH and GFAP.4.The cell growth of DA neurons was most vigorous from the 7^thday to 11^thday of in vitro culture.RT-PCR showed DA neuron-specific genes including Nurr1,lmx1b and TH expressed in the cultured cells.Immunocytochemistry showed the percentage of TH positive neurons was as high as 85.7%.5.Results of APO induced rotation shows 39 of 70 rats（55.7%）were successfully established PD rats.After cell transplantation,behavior test showed cell transplantation can significantly amelioration abnormal rotational behavior induced by APO in PD rat.Compared with other groups,transplantation of GDNF gene modified mNSCs combination with DA neurons has the most striking therapeutic effect.The rotation number of rats in GDNF gene modified mNSCs and DA neurons cotransplantation group decreased nearly 80%compared with before transplantation （P＜0.01）.6.MR images（T2W/FFE）showed that a dark signal appeared in the transplantation area,which gradually became enlargement as time went on.Immunoftuorescence histochemistry showed almost all cells in each group were stayed in situ,not migrating out of transplantation area.Most mNSCs kept undifferentiation state or differentiated into glia cells,only very few cells can differentiate into DA neurons. Compared with other groups,there were more mNSCs differentiated into DA neurons in GDNF gene modified mNSCs and DA neurons cotransplantation group.CONCLUSION1.The GDNF gene eukaryotic expression plasmid pEGFPN1-GDNF is constructed and GDNF gene modified mNSCs are established successfully.2.SPIO particles can effectively label mNSCs,and MRI detection of SPIO labeled cells is a promising method to analysis the cells following intracerebral transplantation.3.Compared with single transplantation of DA neurons or mNSCs,GDNF gene modified mNSCs and DA neurons co-transplantation can significantly improve abnormal behavior of PD rats.However,further research is needed to explore the mechanism of cell transplantation for PD.更多还原