【作者】 董云玲；

【导师】 高英茂；

【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】 干细胞(stem cell)能够不断进行自我更新,具有不同的分化潜能,在特定的条件下,可以分化为不同类型的具有特征性形态、特异分子标志和特殊功能的细胞。根据分化潜力的不同,干细胞可以分为全能干细胞(totipotential stemcell)、多能干细胞(multipotential stem cell)和单能干细胞(unipotential stem cell)。根据细胞来源的不同又可分为胚胎干细胞和成体干细胞。来源于囊胚内细胞团的胚胎干细胞(embryonic stem cell,ES)可以形成除了滋养层细胞以外的所有类型的细胞,进一步形成血液、神经等系统的干细胞,如造血干细胞(hematopoieticstem cells,HSC)和神经干细胞(neural stem cells,NSC)等,它们存在于多种胚胎和成体组织中,在组织修复和细胞数目的动态平衡中起重要作用。近年来,干细胞研究一直处于生命科学研究的前沿位置。干细胞具有的多项分化潜能和自我更新能力使其成为重要的种子细胞,在各种因素导致的组织和器官缺损性、退行性疾病以及先天缺陷性疾病的细胞替代治疗中具有广阔的应用前景。神经干细胞(neural stem cells,NSCs),是一种组织特异性干细胞,能自我更新、增殖,并具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。目前人们已从胚胎、新出生动物和成年动物的小脑、大脑皮层、纹状体、室下区、海马及脊髓等部位成功地分离培养出了这种细胞。近年来,神经干细胞或者整合了特定基因的神经干细胞移植治疗神经损伤和神经退行性疾病的研究取得了令人瞩目的进展。来源于大脑皮层的神经干细胞具有较强的分化潜力和增殖能力,并且数量多、易分离提取,在体内外不同的微环境中可以被诱导分化为具有特定递质类型的神经元,从而可用于细胞替代治疗。肠神经系统(enteric nervous system,ENS)是存在于肠壁的相对独立的神经调节网络,调节消化管的吸收、分泌和运动功能。肠神经节中的神经细胞和神经胶质细胞由神经嵴细胞迁移、分化而来。神经节细胞和神经纤维在肠壁环形肌与纵形肌之间形成肌间神经丛,在粘膜下形成粘膜下神经丛。如果神经嵴细胞不能正常迁入原始消化管,或者迁入的神经嵴细胞不能分化为神经节细胞,就会引起诸多消化道神经运动障碍性疾病,如无神经节细胞性巨结肠病就是这样一种先天性疾病。无神经节细胞性巨结肠病(aganglionic megacolon)又称Hirschsprung氏病(Hirschsprung’s disease,HD,),是一种严重危害婴幼儿健康的先天性畸形,发病率居消化道畸形第二位,据统计高达1/3000～1/5000。其主要病理变化是一段结/直肠肠壁内无神经节细胞,该段结/直肠持续性痉挛,肠腔狭窄,而其邻近的近端肠腔则扩张,内容物潴留。HD主要临床表现是腹胀,顽固性便秘,不完全性肠梗阻。手术切除病变狭窄肠段并进行端一端吻合是目前临床上唯一有效的治疗方法,但治疗效果并不令人十分满意。由于手术损伤和结肠缩短,术后常出现小肠结肠炎、稀便、患儿心理障碍等后遗症,严重者可致结肠破裂。如果病变累及直肠下段或大段结肠,手术切除后,还会引起大便失禁和代谢紊乱。干细胞理论和干细胞移植技术的进展,为该病的治疗提供了新思路,展现出一条崭新的途径。基于上述原因和理念,我们设计了该项研究,目的是为了探讨神经干细胞治疗无神经节细胞性巨结肠病的可行性,为此类疾病的治疗开辟一条新途径。本研究由三部分组成,即无神经节细胞性巨结肠病动物模型的建立、神经干细胞的分离培养和纯化扩增、神经干细胞的肠壁内移植和移植后检测。第一部分无神经节细胞性巨结肠病动物模型的建立进行神经干细胞移植治疗无神经节细胞性巨结肠病的实验研究,首先必须建立一种适宜的该病动物模型。我们根据无神经节细胞性巨结肠病的病理变化特征及原理,以大鼠为实验动物,并根据阳离子表面活性剂苯扎氯胺(benzalkoniumchloride,BAC)能选择性地杀灭神经细胞而不损伤其他组织细胞的原理,经多次实验,找到了使用该药的最佳方式、最佳剂量和最佳作用时间,去除了直肠上段肠壁内肠神经丛的的神经节细胞,而肠壁的其他组织细胞未受损伤。术后4w,肉眼观察发现,BAC作用节段的肠管痉挛、狭窄,其上方肠管则扩张明显、内容物潴留。组织化学、免疫荧光染色等检查证实,BAC作用段肠壁内的肌间和粘膜下神经节细胞消失,神经纤维明显减少,乙酰胆碱酯酶的活性明显降低;直肠内测压显示直肠抑制反射(rectoanal inhibitory reflex,RAIR)消失。通过局部应用BAC的方法,我们成功地建立了无神经节细胞性巨结肠病的大鼠模型,为进一步研究该病的治疗方法奠定了基础。第二部分神经干细胞的分离培养和纯化、扩增进行治疗性干细胞移植实验研究的另一个关键问题是选择理想的和足够数量的供体细胞。由于胚胎大脑皮层中的神经干细胞数量多、易提取、抗原性弱等优点,我们采用了大鼠胚胎大脑皮层来源的神经干细胞,并对其提取、分离时间、培养纯化、扩增方法进行多次实验优化。从孕16天的Wistar大鼠胚胎中分离大脑皮层组织,经胰蛋白酶消化结合机械吹打获得单细胞悬液。用添加B27、表皮生长因子(epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basicfibroblast growth factor,bFGF)的无血清DMEM/F12培养基进行培养。培养72h后开始出现许多由十几个细胞聚集形成的集落样的小细胞团,呈悬浮状态。第6d-7d每个集落的细胞增至几十甚至上百个,形成神经球(neurosphere)。继续培养,每5-7天传代一次。然后通过巢蛋白Nestin的免疫细胞化学染色以检测神经干细胞及其纯度。用含10%胎牛血清的培养液培养进行诱导分化培养,培养7天后的细胞进行MAP2(microtubule associated protein 2)、GFAP(glial fibrillaryacidic protein)免疫细胞化学染色,以检测神经干细胞向神经元细胞和神经胶质细胞分化的特性。结果显示,原代培养和传代培养的细胞球内大多数细胞均呈Nestin强阳性着色。而在含血清培养基中培养7d后可见大量细胞从神经球中迁出,并呈神经元样及胶质细胞样形态,免疫细胞化学染色显示,MAP2阳性细胞占细胞总数的40.64%,GFAP阳性细胞占细胞总数的55.32%。以上结果显示培养的细胞具有神经干细胞的分化潜能。为了检测细胞的增殖情况,我们进行了5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU)掺入实验。结果表明,神经球中有近90%的细胞呈BrdU阳性,这说明培养中的NSCs具有较强的增殖能力。第三部分神经干细胞肠壁内移植和移植后检测在成功建立无神经节细胞性巨结肠动物模型以及成功分离培养、纯化扩增了大鼠胚胎大脑皮层来源的神经干细胞的基础上,我们进行了神经干细胞移植治疗无神经节细胞性巨结肠病的实验。取已预先标记的第5代的神经干细胞,移植于用化学方法去除了神经节细胞的直肠上段。移植后用多种方法检测移植细胞的存活、分化和肠功能恢复情况。结果显示:移植8周后肠壁内有移植细胞存活,并可见部分细胞分化为PGP9.5阳性的神经细胞和GFAP阳性的星形胶质细胞,主要分布于肌间和环形肌层。Western Blot结果表明,BAC去神经节细胞未移植组肠壁内神经型一氧化氮合酶(neuronal nitric oxide sythase,nNOS)及胆碱乙酰转移酶(Choline Acetyltransferase,ChAT)的表达较正常组明显降低,而干细胞移植组在移植后其肠壁内nNOS和ChAT的蛋白表达较未进行细胞移植的模型组明显增强。球囊扩张刺激测压结果显示,去神经节细胞未移植组直肠肛门抑制反射(rectoanal inhibitory reflex,RAIR)消失,而NSCs移植组RAIR恢复;直肠离体肌条的电场刺激(electrical field stimulation,EFS)实验结果也表明,正常对照组和NSCs移植组的直肠肌条在电场刺激(EFS)诱导下均出现较强的收缩反应,而未进行移植的HD模型组则未见明显反应。上述实验结果显示,大鼠胚胎大脑皮层来源的神经干细胞在肠壁内的微环境中可以存活并可分化为功能性神经细胞,从而使肠神经反射和运动功能得到一定程度的恢复。结论本项研究通过建立无神经节细胞性巨结肠病的大鼠实验模型,优化体外分离培养、增殖和纯化大脑皮层神经干细胞的条件,将神经干细胞进行无神经节细胞的肠壁内移植并进行了多项移植后检测,结果证明:取自胚胎大脑皮层的神经干细胞移植到无神经节细胞性巨结肠病动物模型的肠壁中可以存活,并能分化为具有神经节细胞特性的功能性神经细胞,对无神经节细胞性巨结肠有明显的治疗作用。尽管如此,神经干细胞移植的远期治疗效果还有待于进一步研究。更多还原

【Abstract】 Stem cells are specialized cell types in the embryonic and adult tissue.They have the ability of long-term self-renewal and can differentiate into one or more specialized cell types in specific condition.According to their capacity of differentiation,stem cells have been divided into three major groups:totipotent stem cells,multipotent stem cells and unipotent stem cells.The fertilized mammalian egg is totipotent because it can give rise to the entire organism.The inner cell mass of the blastocyst which consists of pluripotent stem cells can give rise to virtually every type of cell except the trophoblast in the body and infinitely proliferate in vitro.These pluripotent stem cells give rise to more specialized cells that are committed to specific lineages such as blood,nervous system,etc.,and are called multipotent stem cells. They are present in many tissues of fetal and adult animals and are important in tissue repair and homeostasis.Good examples include hematopoeitic stem cells(HSC) and neural stem cells(NSC).The ability of multiple lineage differentiation and long term self-renewal make stem cells to be the most important "seed cells" in future regenerative study.Stem cells have come to occupy a permanent important place on the front page for their therapeutic impact.Neural stem cells(NSCs) can be defined as cells which display the ability to self-renew and give rise predominantly to neurons and glia(astrocytes and oligodendrocytes).NSCs have been identified and isolated from various sites of fetal, neonatal or adult mammalian animal and human central nervous system(CNS) and peripheral nervous system(PNS),including the cerebellum,cerebral cortex, hippocampus,olfactory bulb,subventricular zone and spinal cord.Since cortex-originated neural stem cells have a very broad developmental capacity and may generate a multitude of cell types,a great deal of interest has focused on the potential therapeutic applications of them and they have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry. The advantages of using embryonic cortex-originated neural stem cells for transplantation in general include that they have been an extensively studied cell type for this purpose,and they are easily isolated experimentally.Many results of previous in vivo transplant studies have demonstrated their good survival and vigorous neuronal differentiation tendency in the host environment.The enteric nervous system(ENS) is a relatively autonomous collection of neurons and supportive cells that is present in the gut wall and regulates the activity of the gastrointestinal tract,including secretion,absorption,and motility.The neural elements of the ENS come into being ganglionated plexuses located within the gut wall in two relatively distinct collections found either submucosally(submucosal plexus) or between the muscle layer(myenteric plexus).The ENS descends from the neural crest.Neural crest cells,migrating on precisely controlled pathways from the neural tube into the developing gut,function as enteric nervous system stem cells and give rise to all neuronal and glial subtypes found in the gastrointestinal tract. Disturbances and disorders occuring during this time and the process would affect the proper formation and/or the normal function of the ENS,and consequently lead to various severe intestinal dysfunctions,e.g.,Hirschsprung’s disease(HD).HD or aganglionic megacolon or aganglionosis,is a congenital defect that affects 1 in 3000～5000 newborns and it results from a congenital absence of ganglion cells(aganglionosis) in avariable length of bowel.Aganglionosis results in an uncoordinated tonic state in the affected the gut segment resulting in the clinical presentation of bowel obstruction as luminal contents fail to progress.So the affected children usually present shortly after birth with symptoms and signs of distal bowel obstruction.Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children.Typical operative findings are of an apparently narrow distal colon/rectum and a massively dilated proximal colon.The treatment of these enteric neuromuscular disorders is far from satisfactory and remains palliative at best.With regard to the treatment of aganglionic gut conditions, surgical intervention of removal of the affected segments is frequently the only option, whereas surgical therapy does not always lead to a complete recovery and restoration of all bowel functions,which often follows by some complications such as constipation,fecal incontinence and most serious enterocolitis and colonic rupture. The shortcomings in surgery have led to a search for new therapies to improve outcomes in HSCR.Theoretically,a real cure will restore or replace missing or dysfunctional neurons with healthy ones.This has led to the hypothesis that it may be possible to use stem cells to form a "new-ENS" in order to normalize function of the gut.Recent data showed us the exciting progress in the use of NSCs as a valuable therapeutic approach for disorders of the enteric nervous system characterized by a loss of critical neuronal subpopulations.NSCs have been identified in both the developing and adult CNS and PNS of rodents and humans.These cells can be grown in culture and display the ability to self-renew,although they are more restricted than ES cells and give rise predominantly to neurons and glia(astrocytes and oligodendrocytes).The cortex-originated neural stem cells therefore have the potential advantage as the desired phenotype.Whether NSCs could survive well and. differentiate in the gut environment has been unclear.In order to investigate NSCs as an in vitro expandable alternative source for cell replacement therapy in HD,it is essential to set up animal models which closely resemble the neuropathological characteristics of this disease.Therefore,we designed this study and the purpose of the study is to explore the potential of NSCs transplantation as a therapeutic strategy for neuronal replacement of enteric nervous system of rectum in aganglionic rat.This study is a series which consists of three parts as follows:establishment the aganglionic megacolon model by using benzalkonium chloride(BAC) to ablation the ganglionated plexuses selectly;isolation,culture,purifying,proliferation of rat NSCs in vitro;transplantation the neural stem cells into the aganglionic rectum and detection the morphological fate of the grafted cells and evaluation of the enteric motility recovery.Part One:Establishment of the animal model for experimental aganglionic megacolonThe ENS is a well-defined system of neurons and glial cells that regulates several aspects of gastrointestinal physiology.Alterations of the ENS due to disease, age,or other conditions lead to impairment of gastrointestinal function associated with serious disruptions of motility,intractable symptoms,and long-term suffering. The lack of effective therapies for these syndromes due to default of ENS has led us to explore the possibilities of novel approaches such as NSCs transplantation. Therefore,establishment an ideal,steady and repeatable aganglionosis animal model is premise of this study.According to the typical pathophysiology of aganglionosis, we applied a cationic surfactant named BAC to the serosal surface of the rat rectum to ablate the ENS selectively.Four weeks after BAC treatment,the portion treated with BAC was found to be narrowed and the proximal segment became markedly dilated. By detailed histological examination,the segment treated with BAC was confirmed to be denervated and lack of acetylcholinesterase activity;whereas normal myenteric and submucous plexuses and acetylcholinesterase activity were observed in the controls.Rectoanal inhibitory reflex(RAIR) was abolished in the treatment group.A narrow aganglionic rectum was produced successfully in the rat by topical application of BAC to ablate enteric plexus.This animal model will provide the basis for further studies on pathophysiology and new strategy of treatment of ENS disorders such as HD.Part Two:Isolation,culture,purification and proliferation of nural stem cells in vitroTo explore the characterization and the effect of transplantation of NSCs for treatment of disorders of the enteric system such as HD,we systematically investigated the isolation,culture conditions,differentiation and labeling of NSCs in vitro.The NSCs were isolated from fetal brain cortex of Wistar rats(E16) and cultured in Dulbecco’s minimum essential medium(DMEM)/F12-based medium, supplemented with B27,the mitogen epidermal growth factor(EGF),and basic fibroblast growth factor(bFGF).The characterization of NSCs and differentiated cells was identified by immunocytochemistry.Their self-renewal capacity was showed by observing the formation of neurospheres and BrdU immunocytochemistry.Cells were grown as free-floating clusters(neurospheres).By 72 hours under non-adherent culture condition,dozens of NSCs had proliferated to form neurosphere-like aggregates.And after cultured for 6-7 days,neurospheres consisted of hundreds of cells developed.The generated neurosphere was characterized by nestin positive.In the presence of serum,lots of cells migrating from neurosphere differentiated into cells expressing neuronal and astrocyte markers and 40.64%of them differentiated into MAP2-positive neurons compared with 55.32%GFAP-positive astrocytes,which indicated their characteristics of stem cell.Cultures were incubated with bromodeoxyuridine(BrdU).After incorporation with BrdU,approximately 90%cells in neurosphere were detected to be BrdU-positive.The results of our study indicated that neural stem cells could be isolated from fetal brain cortex and the cultured cells maintained the characteristics of neural stem cells,including proliferation, self-renewal and multipotency.It was concluded that fetal cortex-originated NSCs might provide a powerful source of cells for experimental and clinical transplantation.Part Three:Transplantation of neural stem cells into aganglionic rectum of ratOn the basis of successful establishment an aganglionosis animal model and culture,expansion of embryonic cortex-originated neural stem cells in vitro,we explored the possibility of grafting neural stem cells into the aganglionic rectum wall as a therapeutic strategy for enteric neuronal replacement.DAPI-labeled embryonic cortex-originated neural stem cells were transplanted into the denervated rectum. After transplantation,differentiation of grafted cells was examined and the intestinal motility and reflex were assessed.Our results indicated that the grafted NSCs could survive in the gut and then they could differentiate into PGP9.5-positive neurons and GFAP-positive glial cells in vivo after 8 weeks of transplantation.Further assessment for neurochemical showed that the protein expression of neuronal nitric oxide synthase(nNOS) and choline acetyltransferase(CHAT) significantly increased. Moreover,the RAIR and electrical field stimulation(EFS)-induced response were observed in grafted group compared with no reaction in denervated group.It was concluded that cortex-originated NSCs could survive and function in the denervated rat rectum in vivo,which indicated that CNS-NSCs might provide a feasible therapeutic option for some disorders of enteric nervous system.ConclusionsIn conclusion,we set up an experimental aganglionosis animal model successfully,and also successfully isolated,cultured,purified,and expanded cortex-originated NSCs in vitro and then transplanted them into the aganglionic rectum.It showed that the engrafted NSCs could survive,differentiate into functional neuronal phenotypes and restore the defaulted function of gut.This study provided the functional evidence that transplantation of NSCs in the gut might be beneficial in the treatment of motility disorders.Although promising,these data of our study are limited in their short-term nature and further investigation are necessary to assess long-term survival of CNS-NSCs in the gut wall and their functional effect.更多还原