大鼠Cajal间质细胞与神经上皮干细胞体外共培养的实验研究

发布时间：2018-07-01 19:03

本文选题：Cajal间质细胞 + 神经上皮干细胞　；参考：《山东大学》2013年博士论文

【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。
[Abstract]:Hirschsprung's disease (HD) is a common congenital malformation which seriously endangering infant health. According to the relevant statistics, the incidence of the disease is up to 1/3000-1/5000.. The main pathological changes are the absence of ganglion cells in the intestinal wall and the decrease of interstitial cells in the Cajal, the persistent spasm of the intestinal segment, the narrowing of the intestinal cavity, and the narrowing of the intestinal cavity. The main clinical manifestations are abdominal distention, intractable constipation, and incomplete intestinal obstruction. At present, HD is a multi factor, multi gene genetic disease caused by the combination of genetic factors and environmental factors. The abnormalities of the genes regulating the development of ganglion cells in the intestinal wall, such as RET, EDNRB, EDN3, and GDNF, may be HD The internal cause of the disease, while ischemia, hypoxia, toxin, and inflammation may be the environmental factors of HD. Studies have shown that the Enteric Nervous System (ENS) can regulate the motor function of the digestive tube, and it is a relatively independent nervous system. The intestinal nervous system is located in the wall of the intestines, mainly by ganglion cells and nerve fibers. The ganglia consists of nerve cells and glial cells, which are migrated and differentiated from the original neural crest cells. In the process of human embryo development, the neural crest cells fail to move into the original digestive tube for some reason or the migratory neural crest cells fail to differentiate into ganglion cells. The.Cajal interstitial cells (Interstitial cell of Cajal, ICC), such as HD, are located around the nerve plexus and near the ring muscles. It is a cell from mesenchyme. Studies have shown that this cell has electrical slow waves, regulates autonomic rhythmic movement of the gastrointestinal smooth muscle and regulates the intestinal nervous system. The absence or reduction of the function of.ICC, such as stimulating and inhibiting neurotransmitter release, may affect the intestinal pacing activities and obstruct the transmission of neurotransmitters, which leads to the disorder of the autonomic movement of the intestines and the unnormal peristalsis of the intestines, thus causing the onset of HD. Thus, the absence of the interstitial cells between the intestinal God ganglion cells and the intestinal wall of the intestinal wall is reduced. Less or less functional is a direct cause of the onset of HD. Based on the above, we have designed the subject to study the isolation and culture of Cajal mesenchymal cells and neuroepithelial stem cells in rats. At the same time, the two cells were co cultured in vitro to explore the interaction of the two cells and for further cell union. The feasibility of transplantation in the treatment of anthetic cytomegaloenteropathy is feasible and provides theoretical basis. The experiment is divided into three parts: the first part of the rat colon Cajal mesenchymal cells isolation and in vitro culture through in vitro cell culture, we can deeply study cell function and cell signal transduction mechanism. In order to better understand the Cajal stroma Cells, we isolated and cultured the Cajal stromal cells from the colon of newborn rats. We successfully isolated Cajal stromal cells by enzymolysis and density gradient centrifugation. Then, in vitro, we used the M199 medium containing stem cell factor (SCF) to culture for.24 hours, we passed the optical microscopy. The growth of ICCs adherent wall can be seen by the mirror. After 72 hours of culture, it is observed that the cell growth is good, the cell body appears shuttle like.5 days, the protuberances from ICCs are elongated gradually, and the protuberances of adjacent cells are close to each other for.7 days. Under the microscope, the protuberances of adjacent ICCs are interwoven with each other to form a network structure after.7 days. Immunocyte staining was carried out and c-kit positive expression of interstitial cells of Cajal could be observed under the fluorescence microscope. Separation and culture of neuroepithelial stem cells (neuroepithelial stem cells, NESCs) in second parts of rats were the basis and prerequisite for our understanding of stem cells. The optimal scheme for isolation and culture of neuroepithelial stem cells was obtained. The neural tube separated from the rat embryo of 11.5 days of pregnancy was mechanically blown to get single cell suspension and inoculated in the serum-free medium DMEM/F12 containing B27 for.24 hours. After.6 days of the nucleus mitotic phase of the neural epithelial stem cells appeared, dozens or even hundreds of them appeared. The colony formed by the aggregation of the cells, that is, the nerve bulb, and the Nesting staining was positive. The culture medium was replaced by a 10%FBS containing DMEM/F12 for 7 days, and the collagen fibrillary acidic protein (glial fibrillary acidic protein, GFAP) and microtubule related protein 2 (microtubule associated protein 2, MAP2) cells were stained. Positive glial cells and MAP2 positive nerve cells. The combined culture of third parts of Cajal stromal cells and neuroepithelial stem cells combined culture of two cells to better observe the interaction between cells. The culture of third generation of NESCs was inoculated to a good growing ICCs, and the culture medium was 10%FBS containing DMEM/F12, every 3 days. The control group was immunofluorescent staining of protein gene product 9.5 (protein gene product 9.5, PGP9.5) after 7 days of combined culture of NESCs and NESCs cultured with 10%FBS containing DMEM/F12. The results showed that the number of positive cells in the combined culture group was significantly higher than that of the single culture NESCs. The NESCs of the joint culture group was NESCs. The neuronal nitric oxide synthase (neuronal nitric oxide synthase, nNOS) immunofluorescence staining, NESCs can be seen to differentiate into nNOS positive neurons. Through the combined culture of ICCs and NESCs for 7 days, c-kit and MAP2 immunofluorescence double staining, can be seen that two kinds of cells interlinked into the network. Conclusion this study successfully found out The effective methods for the isolation and culture of Cajal mesenchymal cells and embryonic neuroepithelial stem cells in the rat colon were used. On this basis, two kinds of cells were co cultured. It was proved that ICCs could promote the differentiation of NESCs into neurons and nerve cells, and form a network form with the differentiated nerve cells. This discovery provides a theoretical basis for the combined transplantation of two kinds of cells in the treatment of enteric nervous system diseases.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.7

【共引文献】