MGE细胞的培养与移植用于nNOS研究

发布时间：2018-05-05 13:08

本文选题：MGE + nNOS　；参考：《南京医科大学》2017年硕士论文

【摘要】：皮层与海马的信息处理依赖于谷氨酸能兴奋性投射神经元与γ-氨基丁酸(GABA)能抑制性神经元之间复杂的相互作用。两种神经元之间协调的相互作用对于维持大脑兴奋与抑制信号之间微妙的平衡关系至关重要,这种平衡受到很多神经调节物质如不同的神经肽、一氧化氮(NO)等的动态调节。平衡的破坏会导致很多病理性的障碍如癫痫、自闭症、精神分裂症等。NO参与了神经传递、突触可塑性、血管舒张、炎症等很多生理过程,是一个非常重要的信号分子,NO产生或释放功能的缺陷会导致神经元死亡以及癫痫等疾病。NO由一氧化氮合酶(NOS)合成,根据最初被发现时所在的部位,NOS又可分为内皮型一氧化氮合酶(eNOS)、神经元型一氧化氮合酶(nNOS)以及可诱导型一氧化氮合酶(iNOS)。在神经元中NO主要由nNOS合成,在幼稚和成熟的海马与皮层中nNOS主要表达在GABA能中间神经元上。GABA能中间神经元主要来源于三个不同部位:中间神经节隆起(MGE)、外侧/尾部神经节隆起(LGE/CGE)以及视前区(POA),每个神经上皮前体细胞部位都会产生不同的中间神经元亚型。通过在转基因鼠上运用遗传谱系追踪技术发现,皮层I型nNOS主要来源于MGE,II型nNOS则是MGE、LGE/CGE以及POA的混合来源,也就意味着皮层大部分的nNOS来源于MGE。MGE细胞移植到成年鼠的脑内可以扩散、迁移并且分化成GABA—主要的抑制性神经递质。很多假说认为移植MGE的前体细胞可以增加局部的抑制,运用于临床治疗具有非常重要的意义。近几年来分离、移植MGE来源的中间神经元前体细胞方面研究有很多,皮质中间神经元的前体细胞移植到一系列的中枢神经系统组织后可以广泛的迁移并且与宿主神经元网络形成突触联系。MGE细胞移植后可以进行整合的能力被广泛运用到细胞疗法上,从而治疗一系列中枢神经系统疾病:癫痫、神经性疼痛、帕金森以及阿尔兹海默病等。因此运用MGE前体细胞进行的细胞移植在治疗神经和精神疾病中是一个非常有潜能的临床途径。恐惧是一种与适应能力相关的基本情绪,是面对外界可知或不可知的威胁时激发的学习能力。适度的恐惧反应能警示动物面对类似于先前经历的有害环境,及时评估潜在的危险并做出保护反应,而病理性恐惧记忆的产生将会导致创伤后应激综合征(posttraumatic stress disorder,PTSD)、惊恐障碍(panic disorder)、恐惧症(phobia)等恐惧相关疾病。基于巴甫洛夫条件反射原理建立的经典的条件性恐惧(Cued-fear conditioning)以及在此基础上发展出的背景关联恐惧(Contextual fear conditioning)是最常见研究恐惧相关疾病的动物模型,广泛应用于恐惧学习和记忆的神经、分子机制研究。海马是大脑边缘系统结构之一,在情感和认知功能中具有重要作用。既往研究表明,海马与PTSD发生、发展及治疗密切相关,海马DG区是认知功能和情绪调控的神经基础,包含DG区的海马三突触通路对于背景关联型恐惧记忆至关重要。由于目前对于nNOS前体细胞来源鲜有报道,且大部分研究着眼于皮层nNOS细胞。因此,我们希望能通过培养MGE细胞来研究nNOS。本课题设计研究三部分内容:1)通过培养不同部位前体细胞来确证nNOS细胞最主要的胚胎来源是MGE,并改进获取MGE的方法,从而获得状态较好的nNOS细胞;2)对nNOS细胞进行体外培养与鉴定,并尝试多种方法来提高其中nNOS细胞的比例;3)将较高nNOS比例的MGE前体细胞体外培养成神经球,并移植到海马DG区,研究nNOS细胞以及GABA能中间神经元在小鼠恐惧记忆获得中发挥的作用。第一部分为了探究nNOS的胚胎来源,结合已有的文献报导以及对不同时间点nNOS阳性率的研究,我们选取了 E12.5的胎仔,在体视显微镜下取其整脑,并于多聚甲醛中固定24h,随后蔗糖梯度脱水6天后进行冠状冰冻切片,并于载玻片上贴片免疫组化,用免疫标记物PAX6、FOXG-1、Meis-2、Nkx2.1分别标记皮层、前脑、LGE和MGE,对E12.5天胚胎各个部位的形态及范围有个确切的认识。此后,我们就进行E12.5天胎仔的原代培养,在体视显微镜下,分离出同一只胎仔的皮层、POA、CGE、LGE和MGE,体外培养10天后,通过细胞免疫组化鉴定其中nNOS细胞的比例,发现MGE部位nNOS细胞比例最高,即确证了 MGE是nNOS细胞主要的胚胎来源。由于MGE细胞获取方法以及分离的时间长短均会影响原代培养后细胞的状态,因此我们希望能够通过改进现有的MGE获取方法,使得原代培养后的细胞状态更好。综合对比多种方法后,我们尝试了琼脂糖凝胶包埋整脑后Chopper冠状切片、缓冲液中分离MGE的方法以及体视显微镜下于缓冲液中直接分离MGE的方法,后者无论是操作时长、细胞状态以及nNOS细胞阳性率均优于前者。因此第一部分的实验,我们确定了 MGE是nNOS细胞主要的胚胎来源,并通过对比改进了 MGE获取的方法,便于之后的研究。第二部分为了了解MGE来源的nNOS细胞的形态和性质,我们进行了E12.5天胎仔的原代培养,10天后通过免疫组化进行了 nNOS细胞鉴定。发现培养的MGE细胞中90%为GAD67+细胞,99.97%为Tuj-1+细胞,大部分的nNOS细胞可以与GAD67共标,但也有部分nNOS细胞是GAD67阴性的。由于MGE细胞大部分是GABA能中间神经元,且在体能分化形成各种亚型,鉴于体外培养,因此我们也进行了其它亚型的鉴定,发现体外培养的MGE细胞中确实有表达生长抑素(somatostatin,SST)、小清蛋白(parvalbumin,PV)、钙结合蛋白(Calbindin,CB)、钙网膜蛋白(Calretinin,CR)等其它亚型的中间神经元。体外培养的nNOS细胞形态多样,大小不一,且同一玻片上免疫组化的荧光强度也有所差别。由于体外培养的nNOS细胞阳性率较低,因此我们尝试了多种方法来提高其中nNOS的比例。结果表明选取MGE不同部位以及神经营养因子(NGF)的诱导并不能提高nNOS细胞的阳性率。而在不同胚胎期(E11.5-E14)分离MGE,胚胎以及MGE的大小和形态有很大的差别。其中nNOS比例确有差异,E12.5天阳性率最高,其次是E13天。结果表明MGE来源的nNOS经过体外培养,其细胞形态以及性质与体内培养差异不大,且E12.5天获取的MGE细胞nNOS比例最高。第三部分为了进一步探索MGE来源的nNOS在小鼠背景关联型近期恐惧记忆获得中所扮演的角色,我们首先将体外培养3天成神经球的GFP鼠的MGE细胞浓缩,并通过玻璃导管注射到野生型鼠海马DG区,移植2个月后与注射GFP鼠的MGE死细胞相比较,发现野生型鼠中移植MGE细胞并没有恐惧记忆获得值的改变。接下来,我们又将GFP鼠MGE神经球移植到nNOS--鼠海马DG区,与注射死细胞相比,发现nNOS--鼠移植GFP+MGE细胞后,其背景关联型近期恐惧记忆获得显著提高,且nNOS-/-鼠不论雌雄。以上结果表明,野生型鼠海马DG区移植MGE细胞,背景关联型近期恐惧记忆造模后,其恐惧获得的学习能力无改善,而nNOS-/-鼠海马DG区移植MGE细胞,造模后,不论雌雄,其获得恐惧记忆的学习能力提高。为了进一步探究MGE细胞对nNOS-/-鼠行为学的改善作用是否由MGE来源的nNOS细胞主导。我们取B6或nNOS-/-鼠的MGE细胞,体外培养3天成球后浓缩液转染GFP病毒1.5小时,然后悬瓶培养4-6天后,将神经球移植到nNOS-/-鼠海马DG区。2个月后,发现移植B6和nNOS--鼠的MGE对比,nNOS-/-鼠恐惧记忆获得值没有改变,且与宿主的性别无关。为了确证,我们又选取了雌性nNOS-/-鼠进行分析,结果表明nNOS-/-鼠移植B6鼠的MGE与移植死细胞相比,获得值增加,nNOS--鼠移植nNOS-/-鼠的MGE与移植死细胞相比,获得值也增加,但nNOS-/-鼠移植B6或nNOS-/-鼠的MGE细胞之间无差异。以上结果表明nNOS-/-鼠移植MGE后恐惧记忆学习能力的提高源于MGE细胞中非nNOS的其它GABA能中间神经元。结论:(1)MGE是nNOS细胞主要的胚胎来源,并通过对比改进了 MGE获取的方法,便于之后的研究(2)MGE来源的nNOS经过体外培养,其细胞形态以及性质与体内细胞差异不大,且E12.5天获取的MGE细胞nNOS比例最高。(3)野生型鼠海马DG区移植MGE细胞,背景关联型近期恐惧记忆造模后,其恐惧获得的学习能力无改善,而nNOS-/-鼠海马DG区移植MGE细胞,造模后,不论雌雄,其获得恐惧记忆的学习能力提高。nNOS-/-鼠移植MGE后恐惧记忆学习能力的提高源于MGE细胞中非nNOS的其它GABA能中间神经元。
[Abstract]:The information processing of the cortex and hippocampus depends on the complex interaction between glutamate excitatory and gamma aminobutyric acid (GABA) suppressor neurons. The coordinated interaction between the two neurons is essential to maintain a delicate balance between the brain's excitatory and inhibitory signals, which is affected by many nerves. Regulatory substances such as different neuropeptides, nitric oxide (NO) and other dynamic regulation. Balance damage can lead to many pathological disorders such as epilepsy, autism, schizophrenia, etc..NO participates in many physiological processes, such as neurotransmission, synaptic plasticity, vasodilatation, and inflammation, is a very important signal molecule, NO production or release function. Defects, such as neuronal death and epilepsy, are synthesized by nitric oxide synthase (NOS), and NOS can be divided into endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), according to the site where it was originally found. In neurons, NO is synthesized mainly by nNOS in the neuron. In the neuron, the.NO is synthesized in the neuron. The immature and mature hippocampal and cortex nNOS are mainly expressed in the middle neurons of the GABA energy intermediate neurons mainly from three different sites: the middle ganglion bulge (MGE), the lateral / tail ganglion bulge (LGE/CGE) and the preoptic region (POA), and each of the neurons of the preoptic nerve progenitor cells produce different intermediate neurons. By using genetic lineage tracking technique on transgenic mice, the cortical I type nNOS is mainly derived from MGE, and II nNOS is a mixed source of MGE, LGE/CGE, and POA, which means that the majority of the nNOS originates from the transplantation of MGE.MGE cells to the brain of adult mice, and migrates and differentiates into GABA major inhibitory gods. There are many hypotheses that transplantation of MGE's precursor cells can increase local inhibition and is of great significance in clinical treatment. In recent years, there have been many studies on the transplanting of MGE derived intermediate neurons in the precursor cells, and the precursor cells of the cortical neurons were transplanted into a series of central nervous system tissues. The ability to undergo extensive migration and to form synaptic connections with the host network of.MGE cells can be widely applied to cell therapy to treat a series of central nervous system diseases: epilepsy, neuropathic pain, Parkinson and Alzheimer's disease, and so on the use of MGE precursor cells Transplantation is a very potential clinical pathway in the treatment of neurological and mental diseases. Fear is a basic emotion associated with adaptation. It is a learning ability that is stimulated in the face of an outside known or unknowable threat. A moderate fear response can warn the animal to face the harmful environment similar to the previous experience and assess the potential in time. Dangerous and protective reactions, and the formation of pathological fear memory will cause posttraumatic stress disorder (PTSD), panic disorder (panic disorder), phobia (phobia) and other phobia related diseases. The classical conditioned fear based on Pavlov conditioned reflex (Cued-fear conditioning) is based on the classical conditioned fear (Cued-fear conditioning). Contextual fear conditioning, developed on this basis, is the most common animal model for studying fear related diseases. It is widely used in the study of nervous and molecular mechanisms of fear learning and memory. The hippocampus is one of the structure of the cerebral marginal system and plays an important role in emotional and cognitive functions. Previous studies have shown that The hippocampus is closely related to the occurrence, development and treatment of PTSD. The hippocampal DG area is a neural basis for cognitive and emotional regulation. The hippocampal three synaptic pathway, including the DG region, is essential for the background associated fear memory. Since there are few reports on the source of nNOS precursor cells, and most of the studies focus on the cortical nNOS cells. We hope to study the three parts of the design and research of nNOS. based on the cultivation of MGE cells: 1) to confirm that the most important embryonic source of nNOS cells by cultivating different parts of the precursor cells is MGE, and improves the method of obtaining MGE to obtain the better nNOS cells; 2) to culture and identify the nNOS cells in vitro, and to try a variety of methods. To improve the proportion of nNOS cells; 3) the MGE precursor cells of higher nNOS ratio were cultured into nerve spheres in vitro and transplanted into the hippocampal DG region to study the role of nNOS cells and GABA intermediate neurons in the acquisition of fear memory in mice. The first part was to explore the embryonic fetal origin of nNOS, combined with the existing literature and the difference. In the study of the time point nNOS positive rate, we selected the E12.5 fetus, took the whole brain under the stereoscopic microscope, and fixed the 24h in the paraformaldehyde, then the coronary frozen section was followed by 6 days after the sucrose gradient dehydration, and the patch was immunohistochemistry on the slides. The immune markers, PAX6, FOXG-1, Meis-2, and Nkx2.1 were used to mark the cortex, the forebrain, LGE and MGE respectively. There is a definite understanding of the morphology and scope of each part of the E12.5 day embryo. After that, we carried out the primary culture of the E12.5 day fetus. Under the stereoscopic microscope, we isolated the same fetus' cortex, POA, CGE, LGE and MGE. After 10 days in vitro culture, the proportion of nNOS cells was identified by cellular immunization, and nNOS cells in MGE site were found. The highest proportion, that is, confirmed that MGE is the main source of nNOS cells. Because the method of obtaining MGE cells and the length of separation will affect the state of the cells after primary culture, we hope that we can improve the existing MGE methods to make the cells in the primary culture better. The Chopper coronal section of the whole brain after the agarose gel was embedded in the agarose gel, the method of separating MGE in the buffer solution and the method of direct separation of MGE under the stereoscopic microscope in the buffer solution were tried. The latter was superior to the former in the length of operation, the cell state and the positive rate of the nNOS cells. Therefore, the first part of the experiment confirmed that MGE was the main nNOS cell master. In order to understand the morphology and properties of nNOS cells from MGE sources, the second part, in order to understand the morphology and properties of nNOS cells from MGE sources, carried out the primary culture of E12.5 day fetus and identified the nNOS cells by immunohistochemistry. 90% of the cultured MGE cells were GAD67+ cells, 99.97 were found to be GAD67+ cells, and 99.97 were found to be GAD67+ cells. % of Tuj-1+ cells, most of the nNOS cells can be co labeled with GAD67, but also some of the nNOS cells are GAD67 negative. Since most of the MGE cells are GABA in the intermediate neurons, and in the physical differentiation to form a variety of subtypes, in vitro culture, we have also conducted other subtypes of identification, found in the culture of MGE cells in vitro indeed. Expression of somatostatin (SST), parvalbumin (PV), calcium binding protein (Calbindin, CB), calcalum (Calretinin, CR) and other subtypes of intermediate neurons. The morphology of nNOS cells in vitro is diverse and different in size, and the fluorescence intensity of the same slide is also different. In vitro culture of nNOS. The positive rate of cells was low, so we tried a variety of methods to improve the proportion of nNOS. The results showed that the selection of different parts of MGE and the induction of neurotrophic factor (NGF) did not improve the positive rate of nNOS cells. And there was a great difference between the size and morphology of the embryo and MGE in the different embryo period (E11.5-E14) and the size and morphology of MGE. The positive rate of E12.5 days was the highest, followed by E13 days. The results showed that the MGE source nNOS had little difference in cell morphology and nature from the body culture in vitro, and the nNOS ratio of MGE cells obtained on E12.5 days was the highest. The third part was to further explore the short-term fear memory of MGE derived nNOS in mouse background associated type. In order to get the role, we first concentrated the MGE cells of the GFP rat of the 3 day adult nerve bulb in vitro, and injected it into the hippocampal DG area of the wild rat through a glass catheter. After 2 months, we compared the MGE dead cells of the GFP mice. We found that the transplanted MGE cells in the wild mice did not have a change of fear memory. We also transplanted the GFP rat MGE nerve ball into the DG region of the hippocampus of nNOS-- mice. Compared with the injected dead cells, it was found that after the nNOS-- mice transplanted to GFP+MGE cells, the background associated type of fear memory was significantly improved, and the nNOS-/- mice were male and female. The results showed that the MGE cells in the hippocampal DG area of the wild rat were transplanted and the background associated model of the short-term fear memory model was built. The learning ability of the hippocampal DG region of nNOS-/- rats was not improved, and the transplantation of MGE cells in the DG region of the hippocampus of the rat was improved. To further explore whether the effect of MGE cells on the behavioral learning of nNOS-/- rats was dominated by nNOS cells from MGE sources, we took B6 or nNOS-/- mouse MGE cells in vitro. After 3 days of culture, the concentration solution was transfected to GFP virus for 1.5 hours, and then cultured for 4-6 days after suspension bottle culture, after transplantation of the nerve ball to the nNOS-/- rat hippocampus DG area for.2 months, the MGE contrast between the transplanted B6 and nNOS-- mice was found to be unchanged, and it was not related to the sex of the host. In order to confirm, we also selected female nNOS-/- mice. The results showed that the MGE of the transplanted B6 mice of the nNOS-/- mice increased in comparison with the transplanted dead cells. The MGE of the nNOS-- mice transplanted to the nNOS-/- mice increased as compared with the transplanted dead cells, but there was no difference between the nNOS-/- mice and the MGE cells of the B6 or nNOS-/- mice. The above results showed that the learning ability of fear memory in nNOS-/- mice after the transplant MGE was improved. It is derived from other GABA intermediate neurons of non nNOS in MGE cells. Conclusion: (1) MGE is the main embryonic source of nNOS cells, and the method of MGE obtained by comparison is improved to facilitate the subsequent study (2) the nNOS of MGE source is cultured in vitro, and its cell morphology and properties are not very different from the cells in the body, and the nNOS ratio of MGE cells obtained by E12.5 days is obtained. (3) (3) the transplantation of MGE cells in the hippocampal DG region of the wild rat, the learning ability of fear acquisition was not improved after the background related recent fear memory model, and the transplantation of MGE cells in the DG region of the nNOS-/- rat hippocampus, regardless of the male and female, obtained the learning ability of the fear memory to raise the source of the learning ability of the fear memory learning after the.NNOS-/- mice transplanted to MGE. In MGE cells, non nNOS other GABA interneurons.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749

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