【作者】 赵斌；

【导师】 吴荣德；

【作者基本信息】 山东大学 ， 儿科学， 2013， 博士

【摘要】 先天性巨结肠病(Hirschsprung’s disease, HD)是一种常见的、严重危害婴幼儿健康的先天性畸形,根据有关统计,其发病率高达1/3000-1/5000。其主要病理变化是病变肠壁内神经节细胞缺如和Cajal间质细胞减少,病变肠段持续性痉挛,肠腔狭窄,其近端肠腔扩张,内容物潴留；主要临床表现是腹胀,顽固性便秘,不完全性肠梗阻。目前认为,HD是由遗传因素和环境因素共同作用而导致的一种多因子、多基因遗传病。调控肠壁内神经节细胞发育的基因如RET,EDNRB、EDN3、GDNF等的异常可能是HD发病的内因,同时缺血、缺氧、毒素、炎症等可能是HD发病的环境因素。研究证明,肠神经系统(Enteric Nervous System, ENS)能调节消化管的运动功能,并且它是一个相对独立的神经调节系统。肠神经系统位于肠壁内,主要由神经节细胞和神经纤维构成。神经节中包含神经细胞和神经胶质细胞,这些细胞均由原始的神经嵴细胞迁移、分化而来。在人胚发育的过程中,如果由于某种原因造成神经嵴细胞没能迁入原始消化管,或者迁入的神经嵴细胞未能正常分化为神经节细胞,就会引起像HD这样的胃肠道神经运动障碍性疾病。Cajal间质细胞(Interstitial cell of Cajal, ICC)位于神经丛周围及靠近环形肌处,是一种来自间充质的细胞。有研究显示,这种细胞具有产生电慢波,调控胃肠平滑肌自主节律性运动及调节肠神经系统中兴奋性和抑制性神经递质释放等功能。ICC的缺如或减少可影响肠道起搏活动,阻碍神经递质传递,从而导致肠道自主运动功能出现紊乱,肠道不能正常蠕动,从而引起HD的发病。由此可见,肠神经节细胞和肠壁内Cajal间质细胞的缺如、减少或功能异常是HD发病的直接原因。基于上面所述,我们设计了该项研究课题,研究了大鼠的Cajal间质细胞和神经上皮干细胞分离、培养的方法。同时还将两种细胞进行了体外的联合培养,以探讨两种细胞的相互作用,并为进一步的细胞联合移植治疗无神经节细胞巨结肠病的可行性,提供理论依据。实验分三部分进行：第一部分大鼠结肠Cajal间质细胞的分离和体外培养通过体外细胞培养,我们才能深入的研究细胞功能以及细胞信号的转导机制。为了更好地认识Cajal间质细胞,我们对新生大鼠结肠的Cajal间质细胞进行了分离和体外培养。我们采用酶解法和密度梯度离心法,成功分离出了Cajal间质细胞。然后,在体外环境下,用含干细胞因子(SCF)的有血清M199培养基进行进行培养。24小时后,我们通过光学显微镜可以看到ICCs贴壁生长；培养72小时后,可以观察到细胞生长良好,胞体呈现类梭形。5天后,由ICCs发出的突起逐渐伸长,并与周围相邻细胞的突起互相靠近。7天后,显微镜下可以观察到,相邻的ICCs之间突起相互交织,形成网络结构。7天后进行免疫细胞染色,荧光显微镜下可以观察到Cajal间质细胞呈c-kit阳性表达。第二部分大鼠神经上皮干细胞的分离和培养神经上皮干细胞(neuroepithelial stem cells, NESCs)的分离和培养是我们认识干细胞的基础和前提。经过多次试验,我们摸索出了神经上皮干细胞分离培养的最佳方案。将从孕11.5天的大鼠胚胎中分离出的神经管进行机械吹打得到单细胞悬液,接种于含B27的无血清培养基DMEM/F12中进行培养。24小时后,神经上皮干细胞出现核分裂相。6天后,出现几十甚至几百个细胞聚集形成的集落,即神经球,Nesting染色呈阳性。将培养基更换为含10%FBS的DMEM/F12培养7天后,进行胶原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)和微管相关蛋白2 (microtubule associated protein 2, MAP2)细胞染色,结果显示,NESCs分化为GFAP阳性的胶质细胞和MAP2阳性的神经细胞。第三部分Cajal间质细胞和神经上皮干细胞的联合培养将两种细胞进行联合培养,可以更好的观察细胞之间的相互作用。将培养第三代的NESCs接种至生长良好的ICCs上,培养液为含10%FBS的DMEM/F12,每3天换液。对照组为用含10%FBS的DMEM/F12培养的NESCs.将经过联合培养7天的NESCs和单独培养的NESCs进行蛋白基因产物9.5 (protein gene product 9.5, PGP9.5)免疫荧光染色,结果发现联合培养组的阳性细胞数明显高于单独培养的NESCs 。将联合培养组的NESCs进行神经型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)免疫荧光染色,可以见到NESCs分化成nNOS阳性的神经元。将经过联合培养7天的ICCs和NESCs进行c-kit和MAP2免疫荧光双染,可见两种细胞相互交联成网络状。结论本项研究成功地摸索出了大鼠结肠Cajal间质细胞以及胚胎神经上皮干细胞分离和体外培养的有效方法。并在此基础上进行了两种细胞的联合培养,证明了ICCs能促进NESCs向神经元和神经细胞的分化,并在形态上与分化的神经细胞相互连接成网络状,进而可能参与神经信号的传导。这一研究发现为我们将两种细胞联合移植来治疗肠神经系统疾病提供了理论依据。更多还原

【Abstract】 Aganglionic megacolon, i.e. Hirschsprung’s disease (HD), is a common and functional intestinal obstruction with an incidence of 1/3000-1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia and reduction of Interstitial cell of Cajal along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. HD was a multi-factors and multi-genes genetic disease caused by genetic and environmental agents. Abnormality of genes regulating gangliocytes’development such as RET、EDNRB、EDN3、GDNF maybe the inner causes, and ischemia、anoxia、toxin and inflammation maybe the environmental causes.The enteric nervous system (ENS) is a well-defined system of neurons and glial cells that regulates several aspects of gastrointestinal physiology, including motility and secretion. The ENS descends from migrating neural crest cells and is structured into different plexuses embedded in the gastrointestinal tract wall, including myenteric plexus and submucosal plexus. Disturbances appearing during this time affect the proper formation and/or the proper functioning of the ENS, thus leading to severe intestinal dysfunctions, e.g., Hirschsprung’s disease. Intersititial cells of Cajal (ICCs) of the gastrointestinal tract come from the mesenchymal cells of the embryonic layer in the primitive digestive tract, ICCs are the pacemaker cells of the slow wave movement of the intestinal wall, and participate in the transfer of the excitatory and inhibitory neurotransmitter through the contact with the ganglion cell axons in the intestinal wall. Abnormal deficiency or reduction of ICCs and NESCs maybe the immediate cause of HD.Based on the above concept, we addressed this issue to study the isolation and culture of ICCs and NESCs. In this research, NESCs derived from rat embryonic neural tube and ICCs derived from rat colon were co-cultivated in vitro which was designed to explore the interaction and value of the two kinds of cells in the occurrence, development and treatment of enteric nervous system diseases. This study can be divided into three parts.Part one:Isolation and culture of interstitial cells of Cajal from rat colon in vitroIn order to identify ICCs more accurately from cell morphology, we make much efforts in studying the isolation, culture ICCs from rat colon in vitro. With the enzymolysis method in combination with the density gradient centrifugation technology, the ICCs in the digestive tract can be separated and cultivated in vitro. The antibody of c-kit was used to identify the cultured ICCs. M199 medium contains serum and stem cell factor (SCF) was used for resuspending the cell suspension.After 24 hours, we changed the medium. After 3 days, the cell bodies of cultured ICCs seemed like spindles and stretched out a few axis-cylinders. After 5 days, longer axis-cylinders which form synapse each other were observed. Within 7 days in vitro, connections were formed with widespread snapes.Part two:Isolation and culture of neuroepithelial stem cells from neural tube in vitroIsolation and culture of NESCs are the base of realization of the stem cells. The NESCs were isolated from neural tubes of E11.5d Wistar rats. By microdissection and mechanical trituration, a cell suspension was obtained and resuspended in neurobasal medium contining B27. After 3 days, dozens of NESCs had proliferated to form neurosphere-like aggregates and a neurosphere consisted of hundreds of cells developed after cultured for 6 days. The generated neurospheres were characterized as Nestin positive. In the presence of serum, migrating cells from neurosphere differentiated into glial fibrillary acidic protein(GFAP)-positive astrocytes and microtubule associated protein 2(MAP2)-positive neurons.Part three:Co-culture of interstitial cells of Cajal and neuroepithelial stem cells in vitroNESCs and ICCs were co-cultivated in vitro which was designed to explore the interaction and value of the two kinds of cells in the occurrence, development. The third generation of NESCs were inoculated into the ICCs, which the medium is DMEM/F12 with 10% FBS. The control group is the single culture NESCs that is cultivated with DMEM/F12 containing 10% FBS. Protein gene product 9.5(PGP9.5) immunocytochemical staining revealed that the numbers of positive cells of the co-culture group was obviously more than that of the single culture of NESCs. After 7-day co-culture, NESCs were observed to differentiate into the neuronal nitric oxide synthase(nNOS)-positive neurons. The co-cultured ICCs survived well and connected with differentiated NESCs morphologically. Double immunofluorescence staining for c-kit and MAP2 showed that ICCs interconnected with neurons which differentiated from NESCs.ConclusionsIn summary, we successfully isolated and cultured NESCs derived from rat embryonic neural tube and ICCs derived from rat colon. By the co-culture of the two kinds of cells, we demonstrated that ICCs can promote the differentiation of NESCs into neurons, and interconnect with the differentiated neurons into a network morphologically, and then may participate in transmission of nerval signal. The findings may provide the theoretical basis for the treatment of nervous system diseases through the simultaneous transplantation of NESCs and ICCs.更多还原