【作者】 张伟；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 帕金森病(Parkinson’s disease,PD)是中老年常见的中枢神经系统(CNS)退行性疾病,多巴胺(Dopamine,DA)能神经细胞移植是治疗PD的有效手段之一,恰当并且充足的细胞供体源则是成功施行脑细胞移植治疗PD的关键。神经干细胞(Neural stemcells,NSCs)作为具有多重分化潜能和自我更新能力的高度未分化细胞,被视为脑细胞移植治疗脑疾病的理想供体细胞。但NSCs的分化非常复杂,正常情况下,其分化为DA神经元的比例不超过0.01%。因此,在细胞移植前必须先诱导NSCs定向分化为DA神经元,才能有效纠正PD的症状。研究发现,NSCs的分化受到分化微环境中多种因素的影响。目前,已被证实具有促使NSCs向DA神经元定向分化作用的因子包括:IL-1α,IL-11,白血病抑制因子(Leukemia inhibitory factor,LIF)以及胶质细胞源性神经营养因子(Glial cell-derivedneurotrophic factor,GDNF)。Storch等发现在上述细胞因子混合液的基础上加入纹状体细胞分泌液,人类中脑来源NSCs分化为TH阳性多巴胺能神经元的比例骤升了近6倍,分化细胞具有成熟DA神经元形态。Hitoo等发现,将NSCs移植到以6-羟基多巴胺(6-OHDA)毁损制作的PD模型鼠纹状体内后,DA神经元会更多,细胞更大,突起更长,他们用抗体阻断技术证实,在毁损侧纹状体内GDNF和bFGF的分泌增加,有促DA神经元分化作用。这些研究提示,在纹状体内存在着诱导NSCs向DA神经元定向分化的因子,而以6-OHDA毁损黑质-纹状体通路后,纹状体内这些因子的含量显著增强,或者分泌了新的因子。因此本课题拟通过立体定向方法纹状体内注射6-OHDA制备单侧PD模型,利用抑制性消减杂交(Suppression subtractive hybridization,SSH),比较健侧与毁损侧纹状体细胞之间mRNA的表达差异,分析在6-OHDA毁损后,纹状体分泌因子的变化,构建差异cDNA文库,选取相关基因,研究其对神经干细胞增殖、分化的影响。在第一部分实验中采用立体定向方法纹状体内注射6-OHDA制备PD模型,通过行为学以及高效液相色谱电化学方法进行检测,以期建立PD实验研究所需的稳定、可靠的理想模型,为在PD中进一步观察影响黑质纹状体病理变化的基因筛选及其表达的研究提供实验平台。实验结果如下:1.立体定向纹状体内注射6-OHDA制备的PD模型,其行为学改变呈渐进性过程。于术后两周大鼠出现缓慢旋转,直至术后第4周旋转行为达到7转/分以上,并保持基本稳定至第16周。2.高效液相色谱一电化学法(HPLC-EC)组织匀浆检测非注射侧和注射侧黑质中DA含量由0.79nmol/1下降为0.26nmol/1,大约下降了67%,有显著性差异。其代谢产物3,4-二羟基苯乙酸(DOPAC)含量下降62.5%。3.TH免疫组织化学染色可见TH阳性神经元分布于黑质和腹侧被盖部,其形态为大多角形或锥形细胞,胞浆棕染。半定量分析结果统计后显示:注射生理盐水组大鼠黑质部位左、右两侧TH免疫阳性产物数量无显著性差异;术后4周,6-OHDA注射组大鼠黑质部位左、右两侧TH免疫阳性产物数量存在显著性差异(P＜0.05);6、8、12、16周后,注射组大鼠黑质部位左、右两侧TH免疫阳性产物数量均存在极显著性差异(P＜0.01)。在第二部分实验中,采用SSH技术比较PD模型健侧与患侧纹状体组织细胞间mRNA的表达差异,结合分子克隆和反向Northern杂交技术,构建了差异表达cDNA文库。初步筛选和分析了6个上调表达克隆和6个下调表达克隆。功能上较明确的有:高亲和谷氨酸转运体(Slclal);转铁蛋白受体(YrfR);REST/NRSF相互作用限制结构域蛋白(RILP);重组胰岛素样生长因子(Mtpn);不均一核糖核酸核蛋白A/B(Hnrpab);岩藻糖-1-磷酸鸟苷酰基转移酶(Fpgt);前列腺雄激素抑制信使1(Ciparl);纤连蛋白5(fibulin 5);D123基因产物;白介素22α2受体(I122Rα2)。在第三部分实验中,根据第二部分实验结果,并结合文献报道,构建pEGFP-Slclal真核表达质粒,转染体外培养的神经胶质细胞,提取细胞液,观察细胞提取液对c17.2神经干细胞增殖、分化的影响。实验结果如下:1.成功构建了真核表达重组质粒pEGFP-Slclal。2.成功转染pEGFP-Slclal重组子进入培养的神经胶质细胞,可见神经胶质细胞有较多绿色荧光蛋白表达;并且转染pEGFP-Slclal重组子的培养神经胶质细胞中Slclal mRNA呈高表达。3.转染pEGFP-Slclal重组子的培养神经胶质细胞提取液可以提高TH阳性细胞比例,但是与自然状态分化差异不显著(P＞0.05);细胞提取液+细胞因子混合液能明显提高TH阳性细胞含量,与自然状态分化相比具有显著差异(P＜0.01)。总之,在6-OHDA制各单侧PD大鼠模型中,其患侧与健侧纹状体细胞内确实存在多种mRNA的差异表达。对构建的差异表达cDNA文库进行的初步筛选证实了6个上调表达克隆和6个下调表达克隆。对筛选出的12个克隆分析结果提示,PD的发生可能涉及细胞间信息传递、细胞凋亡、细胞内能量代谢障碍、细胞周期调控等多方面因素。对其中的Slclal的研究发现,其可能通过参与维持干细胞的生长、存活,进而影响NSCs的分化、增殖。对其进一步的深入研究,将有助于更好地理解神经干细胞增殖和诱导分化的分子调控机制。更多还原

【Abstract】 Parkinson’s disease（PD） is a common central nervous system degenerative disease happened in middle and aged people.Transplantation of dopaminergic neuron is one of the effective measures in treating PD,which requires suitable and sufficient dopaminergic neuron cell supply.Because of the multiple differentiation potency and self-renewal ability, neural stem cells（NSCs）-highly nondifferentiated cell-are regarded as ideal donorcells in treating cerebral diseases by cell transplantation.However,normally only less than 0.01% of NSCs were differentiated into dopaminergic neuron cells.Therefore,in order to correct the symptoms of PD effectively,NSCs must be oriently differentiated into dopaminergic neuron cells before transplantation.Some studies found that the differentiation of NSCs was affected by many factors in the differentiation microenvironment,which included IL-1α,IL-11,Leukemia inhibitory factor（LIF）,and Glial cell-derived neurotrophic factor（GDNF） currently.Stoch et.al found that by adding secretory fluid of striatum cells to the mixture of above-mentioned factors, The differentiation proportion of NSCs which were originatied from human mesencephalon into TH positive dopaminergic cells was increased by 6 times,and the differentiated cells demonstrated mature shape of dopaminergic neuron cells.Hitoo et.al showed that NSCs differentiated into dopaminergic neuron cells with much higher number,bigger cell body and longer overshoot if they were transplanted into the striatum of PD rats made by 6-OHDA injection.Furthermore,they confirmed that more GDNF and bFGF were secreted from the damaged striatium compared with the contralateral control striatum using antibody blocked technique.All these studies indicated that some factors which can induce oriented differentiation of NSCs into dopaminergic neurons exist in striatum,and if the substantia nigra pathway was damaged by 6-OHDA,the contents of those factors will increase or some new factors may be released.In this study,a mono-side PD model was made by injecting 6-OHDA to the striatum, then the expression difference of mRNAs between health-side and damaged side was compared to analyze the change of secretory factors from the striatum after 6-OHDA damage.A differentially expressed cDNA library was built based on the results,from which several genes were selected to investigate their influence on the differentiation and proliferation of NSCs.In the first part of this study,we tried to build a 6-OHDA induced PD rat model and evaluated it using ethological,high performance liquid chromatogram and electrochemical methods（HPLC-EC）.The aim was to provide a stable and reliable model for further screening of genes which influences the pathological process of nigra-striatum.The results are as follows:1.After injecting of 6-OHDA to the striatum of rats,the ethological changes appeared in a gradual manner.Slow-rotation emerged two weeks after injection and reached 7 turns/min at the fourth week,then kept this condition till the 16^th week.2.The contents of DA and DOPAC,the metabolite of DA,detected using HPLC-EC methods showed that in the 6-OHDA injected side nigra tissue,DA decreased by 67%,from 0.79nmol/l to 0.26nmol/l,compared with that of the contralateral side,and DOPAC decreased by 62.5%.In the striatum tissue DA decreased by 86%,and DOPAC by 94%.3.The TH immunohistochemistry results showed that TH positive neurons distributed in the nigra and ventro-tegmentum area,which was in big polygon or cone shape,and the kytoplasm was brown.The semi-quantitative statistical results showed that the TH positive substance demonstrated no significant change between left and right nigra in the saline treated rats,but changed significantly 4 weeks after 6-OHDA injection in the 6-OHDA treated rats（P＜0.05）,and this difference became more significant after 6,8,12 and 16 weeks（P＜0.01）.In the second part the expression change of mRNA between health side and damaged-side of striatum was compared using SSH technique,and in combination of molecular clone and northern blot techniques a variant cDNA library was built.6 up-regulated and 6 down-regulated gene clones were selected and functionally analyzed, and genes with definite function included TrfR,RILP,Slclal and Mtpn,Hnrpab,Fpgt, Cipar1,fibulin 5,Il22Rα2.In the third part of this study,a pEGFP-Slcla1 eukaryotic expression plasmid was constructed,and transfected to the glial cells cultured in vitro,then the cell sap was extracted to analyze its influence on the differentiation of C17.2 neuronal stem cells.The results were as follows:1.The recombinant pEGFP-Slcla1 plasmid was constructed successfully.2.The recombinant pEGFP-Slcla1 plasmid was transfected to the cultured glial cells successfully,because after transfection a strong green fluorescent protein was observed. Furthermore,the expression of Slcla1 mRNA was very high.3.The cell sap from glial cells transfected with recombinant pEGFP-Slcla1 could enhance the numbers of TH positive cells,but this showed no difference with natural differentiated cells.In comparison with the normal differentiated cells,adding cell sap and cytokine to the culture medium could increase TH positive numbers by 1.5 times.In conclusion,a host of mRNAs expressed differently between damaged striatum cells and normal striatum cells in the 6-OHDA induced unilateral PD rats.6 up-regulated and 6 down-regulated clones were confirmed by screening and analyzing the differential expression cDNA library which was constructed based on the first-part results,and the down-regulated gene-Slclal could enhance cytokine induced differentiation of C17.2 stem cells into dopaminergic neuron cells.Further studies are required to have a better understanding of the molecular modulating mechanism of the proliferation and differentiation of neuronal stem cells.更多还原