【作者】 吴云成；

【导师】 赵永波；

【作者基本信息】 复旦大学 ， 神经病学， 2006， 博士

【摘要】 核受体相关因子1的功能研究及其调控相关基因的筛选研究背景：多巴胺能神经元主要分布于中脑黑质（SN）和腹侧被盖区（VTA），它在调节大脑多种复杂的生理功能中发挥着重要作用，包括随意运动以及奖赏、成瘾等动机行为，其功能异常与帕金森病（Parkinson disease，PD）和药物成瘾（drug dependence／drug addiction）等多巴胺系统疾病密切相关。从酪氨酸到多巴胺（Dopamine，DA）需要经过两个酶解过程，第一个限速酶为酪氨酸羟化酶（tyrosine hydroxylase，TH），它能将酪氨酸代谢为多巴胺前体L-多巴，后者再经过芳香左旋氨基酸脱羧酶（AADC）代谢为DA。而L-多巴可以透过血脑屏障，并被内源性AADC代谢为DA，从而成为PD的主要治疗药物。核受体相关因子1（nuclear receptor-related factor 1，Nurr1／NR4A2／NOT／RNR-1／HZF-3）是一种转录因子，与NGFI-B、Nor1均属于核甾体／甲状腺激素受体超家族成员，它主要表达于中枢神经系统，特别是中脑黑质、腹侧被盖区以及边缘系统。最近有研究表明，Nurr1作为基因转录调控蛋白对DA能神经元的发育、迁移以及存活起关键作用。中脑黑质内Nurr1缺失或功能改变与PD相关，并与其他一些神经精神疾病如精神分裂症、躁狂、可卡因易感等有关。因此，Nurr1已被认为是PD和药物依赖等疾病的重要候选基因。然而，Nurr1在DA神经元发育分化中的调控机制还不清楚，它对DA能神经元表型标记物酪氨酸羟化酶（tyrosine hydroxylase，TH）的调控作用以及促进DA能神经元的成熟方面仍存在争议。另外，我们的近期研究结果提示，GABA转运蛋白亚型1（GABA transporter-1，GAT1）在吗啡和酒精的依赖机制中可能发挥重要作用，但是其具体机制尚不清楚。已有研究表明Nurr1在酒精和可卡因的奖赏强化过程中起重要作用，因此，我们推测Nurr1可能在GAT1导致的药物成瘾机制中起也起到类似的调节作用。材料与方法：本实验共分三个部分，其中第一、第二部分为体外研究，分别按照如下步骤进行。（1）第一部分分三步进行：首先，利用RT-PCR方法对小鼠Nurr1基因进行克隆，然后将其亚克隆至pEGFP-C1质粒的多克隆位点中；其次，将经过酶切和测序证实正确的Nurr1表达质粒用Lipofectamine^TM 2000转染至MN9D细胞中；最后，提取细胞mRNA和总蛋白并利用半定量RT-PCR、实时定量PCR以及免疫印迹技术分别检测Nurr1和TH mRNA和蛋白的表达。同时，利用倒置荧光显微镜观察MN9D细胞的神经突起生长情况，并进行拍照。（2）第二部分亦分三步进行：首先，选择两段RNA干扰靶序列，合成两对编码发夹siRNA序列的单链寡核苷酸，并将其克隆到pSilenCircle载体中，构建含目的基因片段的重组质粒pSC-N1和pSC-N2以及无关基因作为阴性对照质粒，经过转化、扩增以及纯化后进行酶切和测序鉴定。其次，将经过酶切和测序证实正确的质粒用Lipofectamine^TM 2000转染至MN9D细胞中，并用G418筛选两周；最后，提取细胞mRNA和总蛋白并利用半定量RT-PCR、实时定量PCR以及免疫印迹技术分别检测Nurr1和TH mRNA和蛋白的表达。同时，利用倒置荧光显微镜观察MN9D细胞的神经突起生长情况，并进行拍照。（3）第三部分分两个过程：首先，为了进一步证实GAT1基因同源重组的正确性，利用免疫印迹和免疫组化技术对9只GAT基因敲除小鼠（野生型、杂合子和纯合子各三只）海马GAT1蛋白的表达进行了检测；其次，利用实时定量PCR和免疫组化技术检测9只GAT1基因敲除小鼠（野生型、杂合子和纯合子各三只）纹状体和中脑内Nurr1 mRNA和蛋白的表达变化情况。（4）第四部分主要是利用Affymetrix寡核苷酸芯片来寻找MN9D细胞内受到Nurr1诱导而发生变化的基因。首先，常规培养实验组（Nurr1过表达MN9D细胞）和对照组细胞（用RNA干扰产生的Nurr1下调MN9D细胞），提取各组细胞总RNA并逆转录为cDNA。然后合成生物素标记的cRNA，并片断化。最后杂交、洗脱、染色、芯片扫描并利用GCOS软件进行数据分析。结果：下列分别是三部分实验的结果：（1）酶切和测序鉴定证实Nurr1真核表达载体构建成功，Nurr1过表达MN9D细胞内Nurr1 mRNA和蛋白表达明显升高，同时TH mRNA和蛋白表达也明显增高，而RTH升高程度更为显著。另外，Nurr1过表达MN9D细胞的平均神经突起数目、平均神经突起长度和神经突起总长度均明显增加，提示Nurr1可能诱导DA细胞以神经突起延长的特征的细胞成熟和形态分化。（2）酶切和测序鉴定证实Nurr1和无关基因的shRNA表达载体构建成功；pSC-N1和pSC-N2可特异性抑制MN9D细胞中Nurr1 mRNA的表达，下降率分别为62.3％和45.6％；并能使其下游基因THmRNA的表达明显下调，下降率分别为76.3％和62.6％。同时Nurr1和TH蛋白表达亦明显下调，而且TH蛋白表达的下调更为明显。Nurr1和TH蛋白的下降率分别为57.4％、72.0％以及79.1％、70.1％。而阴性对照质粒和脂质体组对两者的mRNA和蛋白没有抑制作用。Nurr1表达下调后MN9D细胞的神经突起数目和长度有所减少，但是无统计学差异。（3）GAT1基因敲除杂合子和纯合子小鼠海马内GAT1蛋白的表达缺失，而野生型小鼠海马内GAT1蛋白表达正常存在，提示GAT1基因同源重组正确。利用实时定量PCR检测发现，与野生型小鼠相比，杂合子和纯合子小鼠脑纹状体内Nurr1 mRNA表达明显增高（p＜0.05）；与野生型和杂合子小鼠相比，纯合子小鼠中脑内Nurr1 mRNA显著升高（p＜0.01）。免疫组化检测结果提示，与野生型小鼠相比，杂合子、纯合子小鼠纹状体区域Nurr1免疫阳性细胞数显著增多（p＜0.01）。杂合子和纯合子小鼠中脑黑质区域Nurr1免疫阳性细胞数比野生型小鼠稍多，但是无统计学差异（p＞0.05）。另外，纯合子小鼠中脑黑质以及杂合子小鼠纹状体区域Nurr1免疫阳性细胞染色明显比野生型和杂合子要深。（4）基因芯片筛选结果发现，有80条基因表达上调、8条基因表达下调，它们主要编码信号转导蛋白、发育和分化相关蛋白、生物合成以及代谢相关蛋白、参与炎症反应和免疫应答蛋白、凋亡、内质网应激以及细胞周期蛋白等。另外，还有24调节基因编码未知功能蛋白。结论：（1）MN9D细胞内过表达Nurr1能促进TH mRNA和蛋白的表达，并能增加细胞神经突起的延长，提示Nurr1对多巴胺能神经元的发育具有关键作用，同时表明它在维持多巴胺能神经元的成熟中具有重要功能。由于DA能神经元能调节随意运动，其变性会导致PD的发生，因此Nurr1对DA能神经元的这种重要功能提示其在PD的基因治疗中具有潜在价值。（2）经过酶切和测序鉴定正确的pSC-N1、pSC-N2质粒能特异性地抑制Nurr1 mRNA和蛋白的表达，提示本实验成功地构建了Nurr1 shRNA的表达载体。同时，它能使TH的表达显著下调，提示Nurr1对多巴胺能神经元内TH的表达调控起关键作用。因此，Nurr1 shRNA特异性表达载体可能为进一步在体内研究PD以及多巴胺能神经元发育相关基因的功能提供新的有效的研究方法。（3）GAT1基因敲除杂合子和纯合子小鼠脑部纹状体内Nurr1 mRNA以及蛋白的表达增强可能是对GAT1表达缺失后导致的GABA失衡产生的一种保护性效应。Nurr1能加强酒精和可卡因依赖的强化特性中的奖赏机制，因此它可能在GAT1相关的药物成瘾机制中发挥正向调节作用。此外，Nurr1可能是药物成瘾机制中DA系统和GABA系统之间的联络中介。（4）基因芯片结果提示，Nurr1可能参与了上述信号转导、分化与发育、生物合成与代谢等过程。尽管仍需要进一步实验加以证实，但是这些结果为我们提供了深入了解多巴胺细胞内Nurr1及其调控基因功能的线索。更多还原

【Abstract】 Background: Midbrain DA neurons, with their cell bodies localized in substantia nigra and the ventral tegmental area, play critical roles in the central regulation of motor and motivational behaviors. Dysfunction of the dopaminergic system of midbrain may lead to dopaminergic system diseases such as Parkinson disease （PD） and drug dependence （or drug addiction）. DA is generated from the amino acid tyrosine in two enzymatic steps. The first rate-limiting step is mediated by tyrosine hydroxylase （TH）, converting tyrosine to the DA precursor L-3, 4-dihydroxyphenylalanine （L-DOPA）, which is converted to DA by L-amino acid decarboxylase （AADC）. L-DOPA, which passes the blood brain barrier and is metabolized to DA by endogenous AADC, is the main treatment in PD. Nuclear receptor-related factor 1 （Nurr1/NR4A2/NOT/RNR-1/HZF-3） belongs to the family of nuclear receptors also including receptors for steroid hormones, retinoids, vitamin D, and thyroid hormone. Nurr1 is expressed predominantly in the central nervous system, especially in substantia nigra （SN）, ventral tegmental area （VTA）, midbrain and limbic areas. Several studies have indicated that Nurr1 is essential for development, migration and survival of dopaminergic neurons. Defects in Nurr1 or altered expression of the gene in SN have been found in association with PD and certain psychiatric disorders, such as schizophrenia, manic behavior, and predisposition to cocaine addiction. Hence, Nurr1 has been already considered as the candidate gene in the mechanism of PD and drug dependence. However, the role of Nurr1 in development and differentiation of dopaminergic neurons has not been elucidated. Especially, there are some disputes in the regulation of Nurr1 on the transcription of TH and maturation of dopaminergic neurons. In addition, our recently studies have indicated that GABA transporter-1 （GAT1） may play a critical role in morphine addiction and ethanol preference. However, the mechanism of these drugs addiction is still not elucidated. There are some evidences that Nurr1 is important for reward mechanisms in the reinforcement of ethanol or cocaine addiction. So, we propose that Nurr1 might play a same role in GAT1 related drug dependence.Materials and methods: Our study was divided into four parts. The first and second parts were all finished in vitro as following steps. （1） The first part was included in the followingthree steps. Firstly, we cloned the mouse Nurr1 gene by RT-PCR, and then it was been subcloned into multiple cloning site of pEGFP-C1 plasmid. Secondly, after being identified by digestion of restriction enzyme and sequencing, the right clones of Nurr1 expressing vector were transfected into MN9D cells with Lipofectamine^? 2000. Finally, the mRNA and total protein were extracted from the stably transfected MN9D cells, Nurr1 and TH mRNA or protein expression was detected using semi-quantitive RT-PCR, Real Time PCR and Western blot. Meanwhile, the neurite extension of MN9D cells was observed and photographed with inversion fluorescent microscope. （2） The second part included three steps too. Firstly, two target sequences in the middle of Nurr1 gene and one target sequence of unrelated gene as a negative control were selected, and then two complementary 72 mer oligonucleotides of each sequence were synthesized with the optimal 3’ overhanging nucleotides. After annealed, the inserts were ligated into the linearized pSilenCircle plasmid. After transformation and amplification, the vectors were purified and identified by gel electrophoresis with restriction enzyme and sequencing. Secondly, the right clone of the constructed pSC-N1, pSC-N2 vectors and the vector of negative control were transfected into MN9D cells and selected with G418 for two weeks. Finally, Nurr1 and TH mRNA or protein expression in different groups were detected using semi-quantitive RT-PCR, Real Time PCR and Western blot. At the same time, the neurite extension of MN9D cells was observed and photographed. （3） The third part included in the following two parts: Firstly, to identify the correctness of the homologous recombination further, GAT1 protein expression in hippocampus were detected in nine GAT1 knockout mice （wide-type, heterozygous and homozygous are three respectively） using western blotting and immunohistochemistry. Secondly, using Real Time RT-PCR technique and immunohistochemistry method, we observed the changes of mRNA and protein expression of Nurr1 in the stratum and midbrain in GAT1 knockout mice （wide-type, heterozygous and homozygous are three respectively）. （4） The fourth part was employed by using Affymetrix oligonucleotide microarrays to investigate changes in gene expression induced by Nurr1 in the MN9D cells. In this part, MN9D cells of the study group （Nurr1-overexpression MN9D cells） and the control group （Nurr1-downregulated MN9D cells by RNA interference） were cultured and the total RNA was extracted. After reverse transcription, doubled stranded cDNA was synthesized and then was further done to produce biotin-labeled cRNA. The cRNA was fragmented and hybridized. Immediately following hybridization, the probe array undergoes an automated washing and staining protocol on the fluidics station. After that, it was scaned and analyzed with GCOS software.Results: The following are the results of the experiments in three parts. （1） Enzyme digestion analysis and DNA sequencing confirmed that the Nurr1 expressing vectors were constructed successfully. Nurr1 mRNA and protein expression in MN9D cells was significantly increased in Nurr1-overexpression MN9D cells. Meanwhile, the expression of TH mRNA and protein is also increased significantly. Moreover, TH expression was upregulated more significantly. The average numbers, average process length and total process length of the neurite extension increased more significantly in Nurr1 overexpression MN9D cells and it is suggested that Nurr1 could induce cell maturation and morphological differentiation, characterized by neurite extension. （2） Enzyme digestion analysis and DNA sequencing confirmed that the Nurr1 or unrelated gene shRNA expression vectors were constructed successfully. Nurr1 mRNA expression in MN9D cells was specifically suppressed after the transfection of pSC-N1 and pSC-N2, the silencing effect is 62.3% and 45.6% respectively using Real Time RT-PCR. The downstream gene TH mRNA is also suppressed significantly and the silencing effect is 76.3% and 62.6% respectively. Meanwhile, the protein expression of Nurr1 and TH were suppressed too, and also the TH protein expression was downregulated more significantly. The silencing effect of Nurr1 and TH protein is 57.4%, 72.0% and 79.1%, 70.1% respectively. While the negative control and liposome has no effect on the mRNA or protein expression of the two genes. Neurite extension of Nurr1 RNA interference group was shorter but has no statistic significantly. （3） The expression of GAT1 protein is deficient in hippocampus in both heterozygous and homozygous mice, while its expression is normally in wide-type mice; it indicates the correctness of GAT1 homologous recombination. Compared to wide-type mice, Nurr1 mRNA expression in stratum is increased significantly in both heterozygous and homozygous mice （p<0.05）, while Nurr1 mRNA in the midbrain of the homozygous mice is increased more significantly （p<0.01）. Meanwhile, Nurr1 immune positive cells in stratum increased more significantly in both heterozygous and homozygous mice than those in wide-type mice by using immunohistochemistry method （p<0.01）. Although Nurr1 immune positive cells in midbrain increased slightly in both heterozygous and homozygous mice, it has no statistic significantly （p>0.05）. Moreover, the staining of Nurr1 immune positive cells in midbrain of homozygous mice and in stratum of heterozygous mice is extremely deep than other mice. （4） Eighty genes were found to be upregulated and eight genes downregulated in the study. They mainly encode proteins for signal transduction, development and differentiation, biosynthesis, metabolite, inflammation and immune response, etc. In addition, there are 22 genes encode proteins whose function have not yetbeen known.Conclusions: （1） Nurr1 overexpression in MN9D cells could promote the expression of TH mRNA and protein and increase neurite extension of the cells. It suggested that Nurr1 was essential for development of dopaminergic neurons and played a major role in the maintenance of the maturation of dopaminergic neurons. The fact that Nurr1 is important for the development of DA neurons has important implications in the gene therapy of PD, as these cells regulate motor control and their degeneration in PD. （2） Nurr1 shRNA expressing vectors had been successfully constructed and could specifically suppress mRNA and protein expression of Nurr1. Meanwhile it could downregulate the expression of TH more significantly. Therefore, it could be concluded that Nurr1 has a critical role in the transcription of TH in dopaminergic neurons. So, Nurr1 specific shRNA expression vector may provide a novel applicable strategy for the function study of genes associated with PD and the development of dopaminergic neuron. （3） The increasing expression of Nurr1 mRNA and protein in stratum and midbrain maybe a protective responses to disequilibrium of GABA system due to the deficient of GABA-transporter 1. Nurr1 could enhance the reward mechanism in the reinforcement of ethanol and cocaine dependence and it might play a positive role in the GAT1 related drug dependence. Moreover, Nurr1 might be a link between DA system and GABA system in the mechanism of drug dependence. （4） The results indicate that Nurr1 might be involved in the regulation of those signal transduction, development and differentiation, biosynthesis and metabolite, etc. Although it needs to be further studied, the data provides clues for further scrutiny into the role of Nurr1 and its regulated genes in the dopaminergic cells.更多还原