【作者】 刘雁平；

【导师】 李露斯；

【作者基本信息】 第三军医大学 ， 神经病学， 2008， 博士

【摘要】 中枢神经系统(central nervous system,CNS)的损伤修复一直是困扰神经科学工作者的大难题。传统观念认为中枢神经系统一旦损伤便不能恢复。近20年来随着神经科学的发展,尤其是干细胞的出现使人们观念发生改变,逐渐认识到成年CNS再生是由神经元的固有特性(intrinsic properties)和其所处的环境(environmental cues)共同决定的。因此增加受损神经元的再生潜能,改善轴突再生的微环境成为CNS损伤修复的基点。神经损伤后修复的难点之一是神经元再生困难,近年来随着干细胞研究的迅猛发展,组织和细胞移植治疗成为大家最为关注的方法之一。选择合适的种子细胞是细胞移植最核心的问题。目前用于移植的细胞供体主要是胚胎干细胞、神经干细胞(neural stem cells,NSC)、来源于神经系统的具有促神经再生作用的细胞(如神经膜细胞、少突胶质细胞、嗅鞘细胞等),以及其他具有成神经分化潜能的成体干细胞。尽管这些细胞在CNS损伤修复移植治疗中均显示出不同程度的积极作用,但由于细胞来源局限、取材不易、免疫排斥以及伦理道德等诸多方面的问题,临床应用受到很大限制。因此,寻找新的种子细胞来源成为当务之急。皮肤是人体最大的器官,包括来源于三个胚层的多种不同的组织细胞,自我更新和再生修复速度快,是成体干细胞分离和研究的良好对象。真皮多能干细胞(Dermal multipotent stem cells,DMSCs)是一种新近分离成功的具有高度增殖能力和多向分化潜能的间充质干细胞,具有分化为脂肪细胞、肌细胞及神经细胞的能力。相对于其他移植供体细胞而言,DMSCs具有来源丰富、容易获得、可自体移植、具有更少的“谱系倾向性”等优点。避免了胚胎伦理学、来源不足、异体排斥等弊端。更有研究表明,DMSCs移植治疗脊髓损伤时,不仅能外在补充缺失神经元,同时可通过分化产生Schwann cells促进轴突再生及髓鞘化。显示了DMSCs在CNS损伤修复中的良好应用前景,有望成为细胞移植及组织工程的新宠。在神经损伤修复中,除细胞移植外在补充缺失神经元外,利用基因工程方法改善损伤局部微环境是治疗的另一个侧重点。肾母细胞瘤过度表达基因(Nephroblastoma Overexpression gene,NOV)是1991年发现的一种原癌基因,其所编码产物(NOV蛋白)是一种胰岛素样生长因子(Insulin-like Growth Factor, IGF)相关蛋白(IGF-relation proteins, IGF-rPs ),属于IGF超家族的成员。NOV基因是神经系统发育的一个调控因子,与动物CNS的损伤和修复之间存在一定的关系。NOV基因可通过IGF依赖或者非依赖的方式发挥作用,参与CNS损伤后的再生和修复过程。NOV基因的真核表达载体可以表达和释放NOV蛋白,促进内源和外源性NSC向神经元的分化,为CNS再生修复提供合适的微环境。NOV基因及其蛋白对DMSCs是否具有类似作用尚未见相关报道。拟将DMSCs作为种子细胞携载NOV基因进入体内,不但能外在补充CNS损伤中缺失的神经元,同时在体发挥改善损伤局部微环境的生物学效应,结合NOV基因治疗及DMSCs细胞移植治疗双重优势用于CNS的损伤修复,是本实验的基本设计思路。为此本文进行了如下研究:(1)含NOV基因的全长序列的真核表达载体的构建和表达;(2)大鼠真皮来源多能干细胞的分离培养和鉴定;(3)观察NOV基因及其分泌蛋白对DMSCs增殖和成神经分化的影响。通过该项研究为结合NOV基因工程及DMSCs细胞移植用于CNS的损伤修复治疗提供初步离体实验资料。研究的主要结果及结论概述如下:1、利用RT-PCR的产物,采用定向克隆的方法,通过中间载体pMD-19T,成功地将NOV cDNA全长序列克隆到pEGFP-N1真核表达质粒上,构建了pEGFP-N1/NOV融合表达载体,用抗性菌落筛选、质粒酶切鉴定、插入片段序列分析等方法,证明获得的重组质粒完全符合设计要求,为进一步的NOV基因功能研究提供分子平台。2、利用早期贴壁传代培养方法,从新生大鼠真皮中分离培养多能干细胞,该细胞经流式细胞仪检测细胞周期、MTT法测定生长曲线具有良好的自我增殖能力;在一定诱导条件下分化为神经细胞、脂肪细胞及成骨细胞,具有向外胚层及中胚层细胞分化的多能性。由此证实了大鼠真皮组织中存在着间充质干细胞,为干细胞移植治疗提供了又一新的种子细胞来源。3、在构建了NOV基因真核表达载体的基础上,用脂质体转染法首次将该重组质粒转染入DMSCs中,流式细胞仪检测转染效率约30%。经G418筛选阳性克隆、多次传代纯化后用RT-PCR法检测到NOV重组质粒在DMSCs中稳定表达。表明DMSCs可作为NOV基因的细胞载体。4、通过细胞形态学观察、流式细胞仪及MTT法研究NOV基因转染对DMSCs增殖性的影响,发现转染了NOV基因的DMSCs较对照组细胞G0/G1期细胞比例下降,细胞倍增时间提前,增殖能力提高一倍。提示NOV基因能刺激DMSCs的增殖。5、收集培养NOV/DMSCs细胞的条件培养液(NOV-CM),结合细胞形态学变化及免疫细胞化学法,研究NOV基因及其分泌蛋白对DMSCs分化的影响,结果显示NOV-CM可以促进DMSCs向神经元方向分化,分化细胞中神经元比例增高,且细胞突起较对照组明显延长。NOV基因转染亦具有同样的作用。综上所述,我们准确的构建了含NOV基因全长序列的真核表达载体,成功分离、培养并鉴定了新生大鼠真皮多能干细胞。在此基础上,采用脂质体法首次将该重组质粒转染至DMSCs中,并且NOV基因在其中稳定表达。进一步的研究表明NOV基因及其分泌蛋白可以显著提高DMSCs增殖性、促进DMSCs向神经元方向分化并有助于突起形成。该实验结果提示DMSCs不仅可作为一种新的种子细胞来源,且携载了NOV基因的DMSCs具有细胞移植及基因工程双重优势,不但外在补充缺失神经元,同时可改善损伤局部微环境,有望在神经系统损伤修复中发挥作用。更多还原

【Abstract】 The recovery of central nervous system (CNS) after injury is all along a difficult problem that puzzles the neuroscientists. In the past, it was widely accepted that once CNS was injured, nothing could be done. Since recent two decades, numerous studies have been carried out by neuroscientists and it is concluded that CNS after injury can be recovered.The course of recovery based on two factors: intrinsic properties and environmental cues. So, it is aimed to promote the regeneration of neurons and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.In regenerative medicine, some encourage results have been obtained. It is well known that cell replacement therapies show particular promise in the nervous system, and transplanted embryonic stem cells (ESCs) or neural stem cells (NSCs) have been shown to promote functional recovery in animal models, for example, spinal cord injury (SCI). Many stem cells (such as ESCs and NSCs) can be used to treat a lot of neurological diseases. Although the therapeutic potential of such transplants is clear, a number of problems, such as insufficient cell number, ethical issues, immunosuppression, and so on, limit their application in clinic. In this background, a new type of stem cells called dermal multipotent stem cells (DMSCs) was discovered in adult mammalian skin. They can differentiate into cells of both neuroectodermal and mesodermal lineage, including (perhaps not limited to) neurons, glia, osteogenic cells and adipocytes. Compared with the above cells, DMSCs, which own multi-oriented differentiative potential and self-renewal capability, have an accessible, potentially autologous tissue source and can expand in vitro cell culture.So they have important therapeutic implications.Deliveries of therapeutic genes are also new and promising strategies to simulate regeneration and recovery of CNS injury. Nephroblastoma Overexpression gene ,abbreviated as NOV, is a proto-oncogene, which was discovered in 1991. NOV protein, which is encoded by NOV gene, is a kind of insulin-like growth factor (IGF) binding protein (IGFBP), and one of the superfamily of IGF. NOV gene is a factor that regulates development of the nervous system. Recent data indicated that NOV gene can provide a possible agreeable microenvironment in regenerative process by IGF-depedent or IGF-indepedent ways. NOV gene may participate in the processes of regeneration and repair after of CNS injury.DMSCs engineered by NOV gene combines with the therapeutic values of DMSCs transplantation and gene delivery. In order to identify the properties of DMSCs and determinate the effect of NOV gene on the proliferation and differentiation of DMSCs, a series of studies were carried out as follows: (1) Construct pEGFP-N1 vector containing NOV gene complete sequence, and examine its expression; (2) Isolate and identify DMSCs from neonatal rat; (3) Study NOV effects on proliferation and differentiation of DMSCs. We hope that the transplantation material—DMSCs modified by NOV gene may express NOV protein eternally, and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.The main results and conclusions are summarized as follows:1. The NOV gene complete sequence was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into pMD-19T vector (NOV-T), then the NOV fragment ligated into HamH I and Hind III sites of eukaryotic expression vector pEGFP-N1 (NOV-N1). The cloned insert in both NOV-T and NOV-N1 were identified by double digestion of the recombinant plasmid with restriction enzymes Hind III and BamH I and by nucleotide sequences. The results suggested that eukaryotic expression vector containing coding region of NOV gene was successfully constructed. The eukaryotic expression vector containing coding region of NOV gene can provide a strong molecular tool for the study of the effect of NOV gene.2. Dermis of neonatal rat were digested with 0.25% trypsin and cultured for 6h in vitro, then the adherent cells were harvested and subcultured. After the fourth subculture, the cells were used for experiments. Cell cycle was analyzed by measuring DNA content with FAC-Scan flow cytometer and cell proliferation activity was tested by MTT method. Differentiated cells were identified by immunocytochemical ABC technique and specific staining. More than 86% of DMSCs were in the G0/G1 phases and these cells display multi-lineage differentiation potential, producing both neural and mesodermal progeny. The results showed Mesenchymal stem cells with multi-lineage differentiation potential exist in neonatal rat dermis and dermis may be another important source of mesenchymal stem cells.3. The recombinant plasmid of NOV gene was transfected into DMSCs at first time with liposome. The expression of NOV gene was observed under Fluorescent microscope and detected by RT-PCR. The ratio of transfected cells was about 30%. DMSCs modified by NOV gene (NOV-DMSCs) offer a chance to study the function of NOV in vivo and DMSCs could be a novel carrier of the NOV gene.4. In order to compare the proliferative capability with NOV-DMSCs and DMSCs, cell cycle was detected by FCM and cell proliferation activity was tested by MTT method.The results indicated NOV-DMSCs in G0/G1 phase decreased while in G2/M and S phase increased than DMSCs did. Meanwhile NOV- DMSCs had shorter doubing time and faster growth velocity. Statistical analysis demonstrated that NOV-DMSCs had higher proliferative capability than DMSCs did.5. Conditioned medium (CM) from cultured NOV-DMSCs (NOV-CM) was collected, and it markedly promoted proliferation of DMSCs. When DMSCs differentiated, NOV-CM helped the differentiation of DMSCs to neurons, and the percent of neurons increased. While DMSCs modified by NOV gene differentiated, the percent of neurons also increased. This result suggested that both NOV gene and protein can improve the neural-oriented differentiation of dermal multipotent stem cells in rats.In summary, dermis mesenchymal stem cells (DMSCs) were isolated from the neonatal rat skin, cultured and identified by their multilineage differentiation capacity. Eukaryotic expression vector containing coding region of NOV gene was successfully constructed and transfected into DMSCs at first time with liposome. Further sdudies showed that both NOV gene and protein could promote the proliferative capability of DMSCs and help their neural-oriented differentiation. The results suggested that not only DMSCs could be a novel carrier of the NOV gene but also DMSCs engineered by NOV gene could combine the therapeutic values of cell transplantation and gene delivery. The results of this study provided some fundmental information for the clinical application of regeneration and recovery of CNS injury.更多还原