躯干神经嵴干细胞的分离、纯化及其向雪旺细胞分化的实验研究

发布时间：2018-01-15 20:09

本文关键词：躯干神经嵴干细胞的分离、纯化及其向雪旺细胞分化的实验研究　出处：《山东大学》2009年硕士论文　论文类型：学位论文

【摘要】： 周围神经损伤是临床上的常见病、多发病。神经损伤后致使支配区的感觉及运动障碍,常常导致肌肉瘫痪、萎缩,甚至留下终生残疾。寻找比较理想的治疗方法一直是当今创伤外科和康复医学等学科的一个研究热点。近年来,组织工程技术的进展为周围神经损伤的治疗带来了新的希望,种子细胞的优化精选是组织工程技术的关键。雪旺细胞(Schwann cell,SCs)是周围神经系统的主要结构和功能细胞,起源于神经嵴干细胞。周围神经损伤后,它既可引导神经轴索的定向生长,又能分泌多种神经营养因子,改善神经再生的微环境,促进周围神经再生及其功能恢复,是公认的种子细胞。然而自体来源的SCs在适当条件下难以获得大量子代雪旺细胞;异体来源的SCs则存在免疫排斥反应等,因此有必要继续寻找更为有效的种子细胞。神经嵴是脊椎动物胚胎早期从神经管背外侧迁移出来的位于神经管和外胚层之间的两条纵向细胞带。神经嵴细胞能够进行自我更新,并可分化为神经元、神经胶质细胞、平滑肌样细胞等多种类型的细胞,也称为神经嵴干细胞(neuralcrest stem cell,NCSC)。自躯干部神经管区域迁移出来的神经嵴干细胞,即躯干神经嵴干细胞(trunk neural crest stem cell,TNCSC),可分化为组成周围神经系统的绝大部分神经元和胶质细胞,是周围神经系统的原基,将躯干神经嵴干细胞应用于神经组织工程有着良好的应用前景。研究表明,神经嵴干细胞可以向神经谱系、成骨细胞等多细胞系方向进行分化,但是由于神经嵴干细胞对体外培养的要求比较苛刻,且取得纯度较高的细胞存在一定的困难,所以对促使其分化为成熟雪旺细胞的影响因素和机制及雪旺细胞与NCSC之间相互的作用鲜有报道。探索如何获取躯干神经嵴干细胞,并增加其向雪旺细胞分化的数量,为组织工程技术提供合适的种子细胞,从而促进周围神经再生和功能恢复,是目前亟待解决的问题。基于以上理念,我们设计了本项实验:取孕10.5d Wistar大鼠的胚胎神经管分离,利用躯干神经管植块法进行培养、纯化神经嵴干细胞,行p75和nestin免疫细胞化学双重染色鉴定培养的躯干神经嵴干细胞,并对其克隆形成能力、增殖、自然分化等生物学特性进行了初步观察;将传代的躯干神经嵴干细胞置于雪旺细胞条件培养液中培养观察,利用MTT分析、细胞计数、免疫细胞化学染色等方法观察躯干神经嵴干细胞存活、增殖、定向分化为功能性雪旺细胞的情况,旨在通过体外培养获得足够量的雪旺细胞,为细胞移植修复周围神经损伤提供实验依据。本实验结果显示,躯干神经管植块培养贴壁24h后可见细胞自组织块边缘迁出,48h后细胞以神经管组织为中心呈辐射状分布,形态多为梭形,少量呈多角形,胞体丰满,胞核较大,免疫细胞化学染色结果显示,神经嵴干细胞的特异性抗体—低亲和力神经生长因子受体的抗体p75及神经巢蛋白nestin染色阳性,表明培养的细胞为躯干神经嵴干细胞。生长曲线计算结果显示躯干神经嵴干细胞的倍增时间为34.2h,细胞呈克隆性生长;在10%血清诱导下,躯干神经嵴干细胞自然分化为神经元、神经胶质细胞和平滑肌样细胞,免疫细胞化学染色显示细胞分别呈MAP2、GFAP、α-SMA阳性。将传代的躯干神经嵴干细胞球在雪旺细胞条件培养液中培养发现24h后细胞贴壁,约36h可见细胞迁出,部分细胞胞体较饱满,突起细长,大体形态与成熟神经元相似;部分细胞有二个或三个突起,呈梭形,形态类似雪旺细胞。随着培养时间延长,突起束状排列,显微镜观察可见在神经元的轴突局部呈双轨样结构,也有少数多个突起的细胞胞体呈不规则形。对照组中神经嵴干细胞球贴壁较少,细胞生长状况较差,随培养时间延长迁出的细胞逐渐减少,形态变化不大,至培养1周,细胞大多死亡。通过细胞免疫荧光染色和图像分析观察到在雪旺细胞条件培养液中,躯干神经嵴干细胞分化的神经元与胶质细胞比例为0.75:1,可分化为大量的神经胶质细胞。MTT分析显示:躯干神经嵴干细胞在条件培养液中存活及增殖活性显著增加(P＜0.01)。培养4周后发现部分轴突局部出现阳性表达MBP,表明分化形成的神经胶质细胞向成髓鞘雪旺细胞方向分化。结论:本实验成功分离培养了躯干神经嵴干细胞并探讨了其生物学特性,并且在雪旺细胞条件培养液中培养时发现有利于躯干神经嵴干细胞存活和增殖,其分化形成的神经胶质细胞可进一步诱导分化形成成髓鞘雪旺细胞,并表达功能性雪旺细胞的主要蛋白MBP。
[Abstract]:Peripheral nerve injury is a common clinical disease, the incidence of sensory and motor disorders. The innervation area after nerve injury often leads to muscle atrophy, paralysis, and even left permanently disabled. Finding the ideal method of treatment has been a hot research topic of trauma surgery and rehabilitation medicine. In recent years, bring new hope for progress in tissue engineering technology for the treatment of peripheral nerve injury, the scaffold and seed cells is the key technology of tissue engineering.
Schwann cells (Schwann cell SCs) is the main structure and function of peripheral nerve system cells derived from the neural crest stem cells. After peripheral nerve injury, it can guide the directional axonal growth and secretion of various neurotrophic factors, micro environment to improve nerve regeneration, promote the recovery and regeneration of peripheral nerve function is recognized. However, autologous seed cells derived SCs under appropriate conditions is difficult to obtain a large number of offspring of Schwann cells; allogeneic SCs immune reaction, it is necessary to find more effective seed cells.
The neural crest is from early vertebrate embryonic neural tube located between dorsal lateral migration of neural tube and ectoderm cells with two longitudinal. Neural crest cells are capable of self-renewal and differentiation into neurons, glial cells, smooth muscle cells and other various types of cells, neural crest stem cells (also known as neuralcrest stem cell, NCSC). Since the trunk neural tube regional migration out of the neural crest stem cells, the trunk neural crest stem cells (trunk neural crest stem cell, TNCSC), can differentiate into most of the peripheral nervous system neurons and glial cells of the peripheral nervous system, is the base, the trunk neural crest stem cells applied to nerve tissue engineering has a good application prospect. The research results show that the cells can differentiate into neural lineages and neural crest stem cells, osteogenic differentiation of multicellular system direction, but due to the neural crest Stem cells require strict culture in vitro, and the cells with high purity have some difficulties. Therefore, there are few reports about the influencing factors and mechanisms that promote their differentiation into mature Schwann cells, and the interaction between Schwann cells and NCSC.
It is a urgent problem to explore how to get the trunk neural stem cells and increase the number of Schwann cells differentiated, and provide suitable seed cells for tissue engineering, so as to promote peripheral nerve regeneration and functional recovery.
Based on the above concept, we design this experiment: isolated from pregnant 10.5d Wistar rat embryonic neural tube, the trunk neural tube explants were cultured, purified neural crest stem cells, p75 and nestin immunocytochemical double staining to identify the cultured neural crest stem cells, proliferation and the colony formation ability, biological characteristics of natural differentiation were observed; the trunk neural crest stem cells into Schwann cells were cultured in liquid culture conditions, using MTT analysis, cell counting, immunocytochemical staining were used to observe neural crest stem cell survival, proliferation, differentiation and function of Schwann cell case and to obtain a sufficient amount of Schwann cells in vitro and to provide experimental basis for cell transplantation in the repair of peripheral nerve injury.
The experimental results show that the neural tube explants cultured 24h cells after the self-organizing block edge to move out, after 48h cells to neural tube tissue as the center of radiate distribution were fusiform, small polygonal, round cell body, larger nucleus, immunocytochemical staining results showed that antibody p75 and nestin nestin neural crest stem - cell specific antibodies of low affinity nerve growth factor receptor staining showed that cultured cells, neural crest stem cells. The results showed that the trunk neural crest stem cell doubling time for the growth curve of 34.2h cells showed clonal growth; in 10% serum induction. The trunk neural crest stem cells differentiated into neurons, glial cells and smooth muscle cells, immunocytochemistry staining showed that the cells were respectively MAP2, GFAP, alpha -SMA positive. The trunk neural crest stem cells. The ball training found that 24h cells adherent fluid in Schwann cells, about 36h cells to move out, some cells are plump, slender projections, gross morphology of mature neurons and similar parts; cells have two or three projections, spindle shaped, with similar morphology of Schwann cells. With longer incubation time. Bundles of axons in the microscope visible part of a track like structure, there are a plurality of protrusions cells were irregular. In the group of neural crest stem cells adherent ball less control, cell growth status is poor, with training time to move out of the cells gradually decreased, morphological changes little, to 1 weeks of culture, most of the cells died. By immunofluorescence staining and image analysis were observed in Schwann cell conditioned medium, the trunk neural crest stem cell differentiation of neurons and glial cell ratio of 0.75:1, can be divided into For a large number of glial cells.MTT analysis showed that the cells increased significantly in survival and proliferation in liquid culture conditions of trunk neural crest stem (P < 0.01). After 4 weeks of culture, found that some local axonal MBP expression, suggesting that glial cells differentiated into medullary sheath Schwann cell differentiation.
Conclusion: the successful isolation and culture of neural crest stem cell and explore its biological characteristics in this experiment, and in the Schwann cell conditioned medium in culture found in favor of the trunk neural crest stem cell survival and proliferation, glial cell differentiation may further differentiate into myelinating Schwann cells, and expression of MBP. protein the function of Schwann cells

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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