【作者】 张丽娟；

【导师】 吴荣德；

【作者基本信息】 山东大学 ， 儿科学， 2014， 博士

【摘要】 背景无神经节巨结肠病即先天性巨结肠病(Hirschsprung’s disease, HSCR),是一种严重危害婴幼儿身心健康的先天性畸形。病变肠段神经节细胞缺失或发育不良,伴随Cajal间质细胞(interstilial cell of Cajal, ICC)缺失或分布异常是其主要病理变化。目前对于先天性巨结肠病的病因和发病机制尚未完全阐明,一般认为该病是由遗传因素和环境因素共同作用而导致的一种先天性遗传疾病。研究表明,肠壁内存在着一个神经调节网络称为肠神经系统(enteric nervous system, ENS),它能相对独立的调节消化系统的运动功能。肠神经系统中的神经元细胞和神经胶质细胞由神经嵴细胞迁移、分化而来。在胚胎发育过程中神经嵴细胞向原始消化管迁入障碍或迁入的神经嵴细胞分化异常均会引起消化管神经运动障碍性疾病的发生。近年来众多的实验研究证实了神经干细胞(neural stem cells, NSCs)移植治疗神经系统退行性疾病和神经损伤性疾病的可行性,提示NSCs移植有望成为治疗消化管神经运动障碍性疾病的新方法。来源于早期神经管壁的神经上皮干细胞(neuroepithelial stem cells, NESCs)除了具有干细胞的基本特征能自我更新和分化之外,还具有取材方便、容易分离培养和扩增的优势以及较低的免疫原性。同时因NESCs具有神经嵴细胞的分化潜能,使其成为细胞移植治疗消化管神经运动障碍性疾病较为理想的供体细胞。在肠道神经丛及肠平滑肌细胞之间还存在一种来源于原始消化管中胚层的间充质细胞,即Cajal间质细胞(ICC)。ICC被称为胃肠慢波活动的起搏细胞,可通过与肠神经丛中的神经节细胞形成突触连接而参与兴奋性及抑制性神经递质的传递。ICC对维持胃肠动力的正常运行至关重要,其缺乏或减少与多种胃肠动力障碍性疾病相关,如先天性巨结肠病、慢传输性便秘、肥大幽门狭窄和假性肠梗阻等。目前ICC已经成为胃肠动力研究的热点,已有研究通过酶解法结合密度梯度离心技术将消化管中的ICC分离出来并进行体外培养,为进一步研究该细胞的功能和相应的细胞信号转导机制奠定了实验基础。鉴于该病主要病理改变是由于神经嵴细胞迁入或分化障碍,以及ICC数量或分布异常所引起,因此从理论上讲,该病理想的治疗方法就是补充缺失的神经节细胞和ICC,即细胞移植治疗。自1978年Sato等首先用苯扎氯铵(benzalkonium chloride, BAC)选择性破坏大鼠肠神经节细胞并成功建立类似于人类先天性巨结肠病的大鼠模型之后,该方法即被广泛应用于该病的实验研究。但关于BAC对肠道内ICC影响的报道甚少。因此本课题在前人研究的基础上,选择利用化学试剂：苄基-二甲基-十四烷基氯化铵(BAC)制作类似于人类先天性巨结肠病的大鼠模型,观察BAC对肠壁内神经节细胞及ICC的影响,并在该大鼠模型上进行NESCs和ICC共移植,检测移植细胞在肠壁微环境中存活、分化和功能状况,并观察细胞移植后的治疗效果,从而进一步揭示先天性巨结肠病的发病机理,并为该病的治疗开辟一条新路。方法本研究通过显微解剖和机械吹打方法分离培养孕11.5天大鼠胚胎神经管来源的NESCs,采用酶消化结合密度梯度离心法自新生Wistar大鼠结肠肌层内分离出ICC,并通过免疫细胞荧光染色的方法对提取的细胞进行鉴定。采用0.5%阳离子表面活性剂BAC选择性地去除大鼠一段降结肠的肠神经节细胞,建立HSCR大鼠模型并在组织学上进行鉴定。分为三组进行移植：NESCs移植组、ICC移植组、NESCs和ICC共移植组。将5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine, BrdU)标记的NESCs和4,6-二氨基-2-苯基吲哚(4,6-diamino-2-phenyl indole, DAPI)标记的ICC移植到HSCR模型大鼠BAC处理肠段。移植后8周,取移植处肠管,利用透射电镜观察移植细胞的存活情况；通过免疫荧光双染和Western blot技术检测肠壁内移植细胞的分化情况。并行离体肌条电场刺激实验和结肠测压球囊扩张刺激反射实验来评估细胞移植后肠运动功能的恢复情况。结果免疫细胞化学染色检测结果显示,NESCs呈Nestin阳性,能分化为微管相关蛋白2(microtubule associated protein2, MAP2)阳性的神经元细胞和胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)阳性的神经胶质细胞。分离培养的ICC呈c-Kit阳性,自胞体发出数条突起,可与邻近ICC突起相互连接形成网络状。BAC作用后4周,PGP9.5、GFAP和c-Kit免疫荧光染色和透射电镜结果均显示模型组BAC处理段肠壁内神经节细胞及ICC消失,而对照组可见正常分布的神经节细胞及ICC。细胞移植后8周,透射电镜结果显示移植细胞存活良好,免疫荧光双染显示移植的NESCs能分化为蛋白基因产物9.5(protein gene product9.5, PGP9.5)阳性的神经元细胞和GFAP阳性的神经胶质细胞,移植的ICC呈c-Kit阳性。NESCs和ICC共移植组移植细胞较单独移植组分化为更多的PGP9.5阳性的神经元细胞(P＜0.05)。Western blot检测显示胆碱乙酰转移酶(choline acetyltransferase, ChAT)和神经型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)在NESCs和ICC共移植组的表达明显升高(P<0.05)。离体肌条电场刺激实验和结肠测压球囊扩张刺激反射实验结果表明共移植组结肠神经运动功能明显恢复。结论本研究在巨结肠病大鼠模型上将NESCs和ICC进行肠壁内共移植,证明了ICC能促进NESCs向肠神经元细胞方向的分化及肠神经运动功能的恢复,证明了肠壁内多种细胞移植治疗先天性巨结肠病的可行性,为细胞移植治疗该病提供了新思路。背景肾母细胞瘤是小儿腹部最常见的恶性实体瘤,其发病率约为0.01%。在过去的数十年中,综合治疗使患儿的长期生存率明显提高,但仍有10%左右的肿瘤患儿因发生复发或转移预后较差。目前关于导致wilms肿瘤转移和复发的主要生物学因素依然未知。近年来的研究表明,炎症在肿瘤发生、发展过程中扮演着重要的角色,其中一些分子机制已逐渐被阐明。肿瘤发生与进展受到多种炎症相关生长因子和细胞因子的影响,包括白介素6(interleukin-6, IL-6)。IL-6通过与靶细胞表面的IL-6受体(IL-6receptor, IL-6R)结合激活IL-6R复合物从而导致下游JAK-STAT3信号通路的活化,促进细胞的增殖、运动和侵袭。本研究拟通过检测小儿Wilms肿瘤组织中IL-6和STAT3(signal transducer and activator of transcription3)蛋白的表达水平评价其与肿瘤侵袭、转移和预后的关系。方法采用免疫组化S-P三步法检测58例原发Wilms肿瘤组织和18例侵袭或转移灶中IL-6与STAT3蛋白的表达水平。结果IL-6在58例Wilms肿瘤原发灶和18例侵袭或转移灶中的阳性率表达分别为39.7%(23/58)和61.1%(11/18)。STAT3在58例Wilms肿瘤原发灶和18例侵袭或转移灶中的阳性率表达分别为29.3%(17/58)和33.3%(6/18)。IL-6和STAT3在侵袭或转移组中的表达高于对照组(P=0.033；P=0.012)。IL-6和STAT3的表达均与病理组织类型相关(P=0.013；P=0.027)。STAT3蛋白的表达差异在各临床分期和患儿年龄间有统计学意义(P<0.05)。在76例Wilms肿瘤组织中IL-6和STAT3蛋白表达具有正相关性(P<0.001,r=0.444)。IL-6和STAT3蛋白在18例原发灶及其对应的侵袭或转移灶中的表达均具有一致性(P=0.727：P=1.000)。IL-6蛋白表达水平和病理组织类型与患儿的无疾病生存期(disease-free survival, DFS)和总生存期(overall survival, OS)相关(P=0.025；P=0.037),而STAT3只与DFS相关(P=0.004),与0S无关(P=0.102)。结论IL-6和STAT3蛋白表达与肿瘤侵袭、转移和预后相关,这为进一步了解Wilms肿瘤浸润和转移的机制及靶向治疗提供新的思路。更多还原

【Abstract】 Background:Hirschsprung’s disease (HSCR) is a serious congenital malformation. It was confirmed that the deficiency or reduced intramural ganglia cells and interstitial cells of Caja (ICC) are the main pathological changes. Currently, the etiology and pathogenesis of this disease have not been fully stated. Although previous studies have showed some genetic factors responsible for human HSCR, other events such as environmental factors have been proved also involved in the incidence of the disease.Studies have shown that enteric nervous system (ENS) was an independent neural regulation system contributed to the alimentary motility. In the process of human development, neural crest cells which contribute to the migration and differentiation of enteric neural cells disabled to move into primitive digestive tube or failed differentiated into neural cells can cause neurological movement disorders of digestive tract. Advances in molecular and stem cell biology have provided new avenues for therapy of ENS disorders. It has been suggested that HSCR could be treated by stem or progenitor cell transplantation to generate a neo-ENS to restore function to the aganglionic bowel. Neuroepithelial stem cells (NESCs), recognized as the most primitive neural stem cells existing in early neural tube wall had the ability of self-renew and differentiated into neurons, astrocytes and oligodendrocytes cells. Moreover, considering the favorableness of isolation, culture, amplification, low immunogenicity and neural crest cell differentiated potential, NESCs have been recognized as the ideal donor for cell transplantation in neurological movement disorders of digestive tube.Interstitial cells of Cajal (ICC) ICC originated from mesodermal mesenchymal cells are the pacemakers in gastrointestinal (GI) muscles which could generate and propagate slow waves and mediate neuromuscular neurotransmission. Evidences showed that absence or reduction of ICC muscles could result in gastrointestinal motility disorders. For now, numbers of researches have separated out the ICC in the alimentary tract through the combination of enzymatic hydrolysis by density gradient centrifugation technology, which facilitated further researched on cell function in vitro.Considering the pathogenesis of HSCR, the ideal treatment is to compensate the missing ganglion cells and ICC, that is, cell transplantation therapy. Since1978, Sato first reported benzalkonium chloride (BAC) could selective destroyed the ENS of rat and successfully established HSCR rat model, the method is widely used for the study of this disease. However, the influence of BAC on ICC is still indistinct. Based on the previous studies, this research aimed to product rat models of HSCR using BAC and observe the effect of BAC on ganglion cells in the colon as well as ICC. Meanwhile, we transplanted NESCs together with ICC to the HSCR rat models to observe the treatment effect of the transplanted cells in intestine micro-environment and evaluate the effect of ICC on neural stem cells after co-transplanted into aganglionic bowel.Methods:NESCs and ICC were isolated from neural tube of embryonic rat (embryonic day11.5) and colon of neonatal rats respectively. Immunofluorescence histochemistry method was used to identify the extracted cells in vitro.0.5%benyalkonium chloride (BAC) was used to damage enteric nervous system to build the rat models of HSCR. After co-transplantation into the BAC-induced rat aganglionic colon, the status of survival and differentiation of the implanted cells were assessed8weeks post-transplantation using immunofluorescence, transmission electron microscope (TME) and western blotting. Colonic motility was assessed by measuring the response of muscle strips to electrical field stimulation (EFS) and the changes of intraluminal pressure responding to inflating stimulation.Results:The isolated NESCs which exhibited the Nestin immunoreactivity could differentiate into neurons and astro-cytes with MAP2and GFAP immunoreactivity respectively. The isolated ICC displayed c-Kit immunoreactivity and could interconnected with adjacent ICC by the multiple processes extended from the cell body. Four weeks after surgery, histological examinations and TEM showed that ganglion cells and interstitial cells of Cajal (ICC) significantly reduced or completely disappeared at the segment of BAC treated sit. Eight weeks after transplantation, the results showed the transplanted cells survived well. The BrdU labeled NESCs could differentiate into PGP9.5positive neurons and GFAP positive glial cells. The DAPI marked ICC with c-Kit immunoreactivity survived well and form connections with the differentiated NESCs. The co-transplantation group showed more differentiated PGP9.5-positive neurons compared with transplantation of NESCs alone (P<0.05). Western Blotting detection showed the neuronal Nitric oxide synthase (nNOS) and choline acetyl transferase (ChAT) expression were markedly elevated in the co-transplantation goup (P<0.05). Moreover, the EFS-induced reaction of the colon and contractile response to inflation after co-transplantation were successfully evoked.Conclusions:Our data indicate that reconstitution of elements in the intestinal environment such as ICC enhances the differentiation of NESCs. This may aid development of a stem cell-based treatment for HSCR. Background:Wilms’tumor (WT) is one of the most common malignant tumors in children with an incidence of approximately1in10000. The prognosis of the patients has greatly improved by the advancement of combination therapy. Nevertheless, about10%patients with WT have poor survival suffering from metastasis or recurrence. Although previous studies have showed some aberrant molecular events responsible for tumor progression, the biological factors governing Wilms’ tumor invasion and metastasis remain largely unknown. Research has suggested that deregulated inflammation is associated with most tumors. Tumorigenesis and progression associated with inflammation are known to be influenced by multiple growth factors and cytokines including interleukin-6(IL-6). The IL-6signaling pathway is activated via binding to its receptor IL-6R resulting in downstream signal transmission through various signal pathways including JAK2/STAT3signal pathway and ultimately leading to induction of target genes that control cell proliferation, tumor invasion and metastasis. This study aimed to investigate the expression profiles of IL-6and STAT3in Wilms’ tumor (WT) and their relationship with disease progression.Methods:Immunohistochemistry was used to examine IL-6and STAT3expression status in58primary tumors and18invasive/metastatic ones.Results:Positive expression rate of IL-6/STAT3was39.7%(23/58)/29.3%(17/58) in primary WT tissues, while61.1%(11/18)/33.3%(6/18) in associated invasive/metastatic tissues. The expression rate of IL-6and STAT3was higher in primary WT tumors of invasive/metastatic group than that of non-invasive/metastatic group (P=0.033; P=0.012). The expression rate of IL-6and STAT3was also associated with histopathological type (P=0.013; P=0.027). There was a positive correlation between IL-6and STAT3expression in76WT tissues (P＜0.001, r=0.444). The expression of IL-6/STAT3between primary WT and matched invasive/metastatic tissues was concordance (P=0.727; P=0.99). IL-6expression status and histopathological type were associated with disease-free survival (DFS) and overall survival (OS)(P=0.025; P=0.037), while STAT3was only correlated with DFS (P=0.004).Conclusions:IL-6and STAT3expression in WT might be correlated with progression and predict unfavorable prognosis, highlighting a new therapy target for invasive or metastatic WTs.更多还原