内皮祖细胞调控神经干细胞分化及其机制研究

发布时间：2018-06-28 19:10

  本文选题：脊髓损伤 + 神经干细胞　； 参考：《第三军医大学》2010年硕士论文

【摘要】： 现代社会文明进步发展的同时,也带来了人类社会第一大公害“创伤”的大量发生,创伤被称为“发达社会病”。脊柱、脊髓损伤(spinal cord injury,SCI)是交通、劳动和运动等意外事故中常见的创伤类型。脊髓损伤是人类致残率最高的疾患之一,一直是困扰医学界的一大难题。它可直接导致损伤平面以下的感觉、运动功能丧失及排尿、排便功能障碍。损伤的平面越高,患者丧失的功能越多。美国有截瘫患者约253000例,每年新增约11000例,每年为这些截瘫患者支付的医疗费用高达60亿美元,而在中国截瘫患者人数约40万,每年新增1万人,给患者、家庭和社会带来巨大的负担。但到目前为止国内外治疗脊髓损伤的药物和外科手术虽均有进展,但却未取得满意的临床疗效。因此,加强SCI救治与截瘫康复的研究具有重要的社会现实需求和科学理论价值。 近年兴起的利用组织工程学的方法来修复脊髓损伤给脊髓损伤患者带来了新的希望。组织工程学是生物医学工程学中的一个新的分支,是应用生命科学和工程学的原理和技术,构建人工组织以修复组织器官的结构和功能。其主要的研究内容包括:种子细胞的筛选、支架材料的合成与塑型以及组织的构建。目前,用于构建脊髓组织工程的种子细胞主要包括:胚胎干细胞、神经干细胞(neural stem cells,NSCs)、骨髓间充质干细胞、嗅鞘细胞、雪旺细胞和成纤维细胞等。种子细胞主要通过以下方式来修复脊髓损伤:1)替代损伤或死亡的细胞;2)桥接脊髓损伤断端,形成功能性突触,重新建立神经传导通路;3)改善临床症状,提供神经保护和生长因子使受损的轴突易于再生;4)起到其他的间接作用:如促进血管生成,为再生和内生细胞提供营养支持。 NSCs被认为是构建脊髓组织工程最佳的种子细胞。NSCs为多能干细胞,终身具有自我更新能力和多向分化潜能,无论是在哺乳动物胚胎神经组织还是成年个体的脑组织中都存在,是中枢神经系统内新生细胞产生的源泉。NSCs在一定条件下能分化为神经元、星形胶质细胞和少突胶质细胞。并且,NSCs具有易于获取、培养方法成熟,鉴定容易,可在体外大量增殖、分化等优点,适合作为组织工程神经移植物实验的种子细胞。尽管从理论上讲NSCs的功能完美无缺,但近年来研究发现单独NSCs应用于成年SCI的治疗存在如下问题:移植后NSCs分化为神经元少,无法提供大量功能性轴突连接所需的神经元,而胶质细胞大量增生,形成的胶质瘢痕不利于轴突再生;由于损伤脊髓局部的血运不能得到及时恢复,变性坏死组织及毒性产物不能很快清除,使移植物中的神经细胞得不到充分的血管营养支持,导致细胞存活时间短,神经干细胞增殖分化困难,达不到应有的治疗效果。因此,如何促进移植到损伤脊髓的NSCs向神经元分化、促进损伤脊髓血管再生以保证移植细胞的血管营养支持,成为利用NSCs及其构建的组织工程移植物移植修复损伤脊髓亟待解决的关键问题之一。 血管内皮祖细胞(Endothelial Progenitor Cells,EPCs)是Asahara等在1997年首次从成体外周血中分离,并证实在人类出生后的外周循环血液中存在,能分化为血管内皮细胞的前体细胞。EPCs是一群具有游走特性,并能增殖、分化为内皮细胞的前体细胞,包括从血液血管母细胞到成熟内皮细胞之间多个阶段的细胞,亦称为血管母细胞或血管内皮干细胞,不仅参与胚胎时期血管发生,而且在出生后成体血管新生过程中也起重要作用。EPCs能迁移并结合到血管再生的部位,增殖、分化为成熟的内皮细胞,参与局部新生血管的形成。EPCs的这些特性使其具有广泛的临床应用价值,可作为组织工程中血管的种子细胞来源。近年来,有关EPCs在组织工程血管再生中作用的研究主要集中在缺血心肌、缺血肢体、损伤皮肤和损伤角膜等方面,还尚未用于脊髓损伤。我们拟选用NSCs和EPCs作为种子细胞,构建血管化组织工程脊髓来修复损伤的脊髓,以同时解决脊髓损伤后的“神经营养”和“血管营养”,这两大问题。然而,NSCs和EPCs来源于体内不同胚层,体内和体外对周围生长的环境要求不同。要同时移植这两种细胞到宿主体内,需先在体外对这两种种子细胞进行共培养以探讨两者最佳的生长条件以及共培养后两者生物学特性的变化规律,从而为构建血管化细胞组织工程移植修复脊髓损伤和中枢神经系统疾病提供可行性实验依据和理论基础。本实验拟从大鼠的外周血中分离单个核细胞、经体外培养、诱导后获得EPCs,在体外与NSCs进行共培养,观察两者生长情况及NSCs的分化方向,然后对其作用机制进行初步探讨。 主要方法和技术路线 实验分为三个部分 1.采用孕龄14d的SD胎鼠大脑皮层组织,分离纯化培养NSCs,血清诱导NSCs分化。分别采用Nestin、β-tubulin-Ⅲ和GFAP对NSCs球、分化后的神经元和星形胶质细胞进行鉴定。采用密度梯度离心法从重约180g的SD大鼠外周血中得到单个核细胞,通过贴壁培养、体外扩增获取细胞,通过观察细胞形态、CD34/Ⅷ因子免疫荧光反应等方法进行鉴定。 2.在体外进行EPC与NSCs共培养,按培养基不同分为NB培养基组、DMEM培养基组和NB+DMEM培养基组,观察两种种子细胞生长情况,以寻找最佳条件培养基。观察EPCs对NSCs增殖和分化的调控作用,并初步探讨两者不同比例情况下,NSCs分化变化规律。 3.初步探讨体外共培养的EPCs调控NSCs分化为神经元的作用机制。ELISA法检测EPCs、NSCs及两者共培养的上清液VEGF、BDNF含量。观察VEGF、bFGF联合应用对NSCs分化的影响,并利用VEGF的抗体进一步探讨VEGF是否在EPCs与NSCs共培养时,促进NSCs向神经元方向分化中起重要作用。 4.统计学分析:各组数据以xv±s表示,运用SPSS13.0进行统计分析,组间比较采用t检验,以P0.05为显著水平,P0.01为非常显著。 主要的研究结果和结论如下: 1.胎鼠大脑皮层NSCs丰富,易于获取,培养的NSCs可在体外扩增,并在一定的条件下能分化为神经元和神经胶质细胞。大鼠外周血来源丰富,取材方便,分离出的EPCs在特定的培养条件下能分化为内皮细胞。提示本实验方法能够体外成功培养出NSCs和EPCs种子细胞。 2. EPCs与NSCs共培养时,最适合的条件培养基为NB+DMEM(1:1)。在此条件下,EPCs能明显促进NSCs的增殖,且调控NSCs定向分化为神经元的比例(68.40%)明显高于单纯血清诱导组(28.7%)。且随着EPCs:NSCs的比例由1:10到10:1,这种分化诱导作用越显著。提示本实验所建立的EPCs与NSCs共培养体系是可行的,且在该体系中EPCs能明显促使NSCs向神经元方向分化。 3. ELISA法检测EPCs及其与NSCs共培养时上清液中的VEGF分别为817.23 pg/ml、917.78 pg/ml,明显高于NSCs单培养时363.67 pg/ml,表明EPCs主要分泌VEGF。 4. VEGF、bFGF诱导NSCs分化为神经元的比率分别为60.3%、60.4%,两者联合应用时神经元分化比率明显提高达80.3%。表明VEGF能明显促进NSCs向神经元方向分化,且与bFGF有协同作用。 5.共培养时EPCs对NSCs向神经元分化的调控作用,能被VEGF抗体明显抑制;而在加入与VEGF抗体等量的VEGF后,EPCs仍然能明显促进NSCs向神经元分化。表明EPCs可通过其分泌的VEGF调控NSCs向神经元分化。
[Abstract]:At the same time, the progress and development of civilization of modern society also brought about the massive occurrence of "trauma" of the first public harm in human society. The trauma is called "developed social disease". Spinal cord injury (spinal cord injury, SCI) is the common type of trauma in traffic, labor and sports accidents. Spinal cord injury is the highest disability rate of human being. One of the problems that has been plaguing the medical community has been a direct cause of sensation below the level of injury, loss of motor function, urination, and defecation dysfunction. The higher the level of injury, the more the patients lose. There are about 253000 paraplegic patients in the United States, about 11000 new cases a year, and the higher medical costs for these paraplegic patients each year. The number of paraplegic patients in China is about 6 billion dollars, and the number of paraplegic patients in China is about 400 thousand, and 10 thousand people have added a huge burden to the patients, family and society each year. However, although there has been progress in the treatment of spinal cord injury at home and abroad so far, however, it has not achieved satisfactory clinical effect. Therefore, the study of strengthening SCI treatment and paraplegia rehabilitation is heavy. The need for social reality and the value of scientific theory.
The use of tissue engineering to repair spinal cord injury in recent years has brought new hope to the patients with spinal cord injury. Tissue engineering is a new branch of biomedical engineering. It is the application of the principles and techniques of life science and engineering to construct artificial tissues to repair the structure and function of tissues and organs. The contents include: screening of seed cells, synthesis and molding of scaffold materials and construction of tissue. At present, seed cells for the construction of spinal cord tissue engineering include embryonic stem cells, neural stem cells (neural stem cells, NSCs), bone marrow mesenchymal stem cells, olfactory ensheathing cells, Schwann cells and fibroblasts. Repair spinal cord injury in the following ways: 1) replace damaged or dead cells; 2) bridging the injured end of the spinal cord, forming functional synapses, reestablishing the nerve conduction pathway; 3) improving the clinical symptoms, providing neuroprotection and growth factors to make the damaged axons easy to regenerate; 4) other indirect effects, such as promoting angiogenesis, and re Raw and endophytic cells provide nutritional support.
NSCs is considered to be the best seed cell for the construction of spinal cord tissue engineering,.NSCs is a pluripotent stem cell. It has a lifelong self renewal capacity and multidirectional differentiation potential, both in the mammalian embryonic neural tissue and in the adult brain tissue. It is the source of the source.NSCs of the new cells in the central nervous system under certain conditions. Differentiation into neurons, astrocytes and oligodendrocytes. Moreover, NSCs has the advantages of easy acquisition, maturation, identification, proliferation and differentiation in vitro. It is suitable as a seed cell for tissue engineering nerve graft experiment. Although the function of NSCs is perfect in theory, it has been discovered in recent years. The treatment of single NSCs for adult SCI has the following problems: after transplantation, NSCs is differentiated into less neurons and can not provide a large number of neurons needed for functional axonal connections, while glial cells proliferate, and glial scar formation is not conducive to axonal regeneration; the blood transport of the injured spinal cord can not be recovered in time, degeneration and necrosis and poison. The sex products can not be removed quickly, so that the nerve cells in the grafts can not get sufficient vascular nutritional support, which leads to the short survival time and the difficulty in the proliferation and differentiation of neural stem cells. Therefore, how to promote the transplantation of NSCs to the injured spinal cord to differentiate the nerve cells and promote the regeneration of the injured spinal cord to guarantee the transplantation of the spinal cord. Cellular vascular nutritional support has become one of the key problems to be solved urgently by using NSCs and its tissue engineered graft to repair injured spinal cord.
Vascular endothelial progenitor cells (Endothelial Progenitor Cells, EPCs) are the first isolation of Asahara from the peripheral blood in 1997, and proved to exist in the peripheral circulating blood after human birth, and.EPCs, a precursor cell that can differentiate into vascular endothelial cells, is a group of precursors that have a wandering, proliferation and differentiation into endothelial cells. The cells from blood vessels to mature endothelial cells, also known as angioblastoma or vascular endothelial stem cells, not only participate in embryonic angiogenesis, but also play an important role in the process of angiogenesis after birth and.EPCs can migrate and bind to the site of vascular regeneration, proliferate, and differentiate into maturity. These characteristics of endothelial cells, which participate in the formation of.EPCs in local neovascularization, have a wide range of clinical applications and can be used as the source of seed cells in tissue engineering. In recent years, the research on the role of EPCs in vascular regeneration in tissue engineering is mainly focused on ischemic myocardium, ischemic limbs, skin injury and injury of cornea. It is not yet used for spinal cord injury. We use NSCs and EPCs as seed cells to construct vascularized tissue engineered spinal cord to repair the injured spinal cord, and to solve the two major problems of "neuronutrition" and "vascular nutrition" after spinal cord injury. However, NSCs and EPCs are derived from different germ layers in the body, in vivo and in vitro The growth environment is different. To transplant the two cells to the host at the same time, the two seed cells should be co cultured in vitro to explore the best growth conditions and the changes of the biological characteristics after co culture, so as to construct the vascularized cell tissue engineering transplantation for the repair of spinal cord injury and central God. This experiment provides the basis and theoretical basis for the feasibility of systemic disease. This experiment is to separate mononuclear cells from the peripheral blood of the rat. After culture in vitro, EPCs is obtained and co cultured with NSCs in vitro. The growth of the two and the differentiation direction of NSCs are observed. Then the mechanism of its action is preliminarily discussed.
Main methods and technical routes
The experiment is divided into three parts
1. the NSCs was isolated and cultured in the cerebral cortex of SD fetal rat of gestational age. NSCs was isolated and cultured to induce the differentiation of NSCs. Nestin, beta -tubulin- III and GFAP were used to identify the neurons and astrocytes after the differentiation of NSCs balls. The density gradient centrifugation was used to obtain mononuclear cells from the peripheral blood of SD rats weighing 180g about 180g, through the adherent culture. The cells were obtained from in vitro expansion and identified by observing the cell morphology, CD34/ VIII factor immunofluorescence reaction and so on.
2. co culture of EPC and NSCs in vitro, divided into NB medium group, DMEM medium group and NB+DMEM medium group according to the culture medium, observe the growth condition of two seed cells in order to find the best condition medium, observe the regulation of EPCs on NSCs proliferation and differentiation, and discuss the differentiation and change regularity of NSCs under the condition of the two different proportions.
3. preliminary study on the role of EPCs in vitro co cultured in vitro to regulate NSCs differentiation into neurons..ELISA method was used to detect EPCs, NSCs and the content of VEGF and BDNF in the co culture supernatant. The effect of VEGF, bFGF combined application on NSCs differentiation was observed. It plays an important role in differentiation.
4. statistical analysis: the data of each group were expressed as XV + s, and SPSS13.0 was used for statistical analysis. The t test was used in the comparison between the groups, and P0.05 was the significant level. P0.01 was very significant.
The main results and conclusions are as follows:
The cerebral cortex of 1. fetal rats is rich in NSCs and easy to obtain. The cultured NSCs can be amplified in vitro, and can be differentiated into neurons and glial cells under certain conditions. The peripheral blood of rats is rich in peripheral blood, and the isolated EPCs can be differentiated into endothelial cells under specific culture conditions. This method can be successfully cultured in vitro. NSCs and EPCs seed cells were produced.
2. EPCs and NSCs co culture, the most suitable conditioned medium is NB+DMEM (1:1). Under this condition, EPCs can significantly promote the proliferation of NSCs, and the proportion of NSCs directed differentiation into neurons (68.40%) is significantly higher than that of the pure serum induction group (28.7%). And with the proportion of EPCs:NSCs from 1:10 to 10:1, the more significant differentiation induction. The co culture system of EPCs and NSCs established in the experiment is feasible. In this system, EPCs can obviously induce NSCs to differentiate into neurons.
The VEGF of the supernatant in the co culture of EPCs and NSCs by 3. ELISA method was 817.23 pg/ml and 917.78 pg/ml respectively, which was significantly higher than that of 363.67 pg/ml during the single culture of NSCs, indicating that EPCs secreted VEGF..
4. VEGF, the ratio of NSCs differentiation into neurons by bFGF was 60.3%, 60.4% respectively. The differentiation ratio of neurons was significantly increased by 80.3%., indicating that VEGF could obviously promote the differentiation of NSCs into the neuron and have synergistic effect with bFGF.
5. in co culture, the regulation of EPCs on the differentiation of NSCs to neuron can be significantly inhibited by VEGF antibody, and EPCs can obviously promote the differentiation of NSCs into neurons after adding VEGF to VEGF antibody. It indicates that EPCs can differentiate into neurons by its secreted VEGF regulation NSCs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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