【作者】 胡亚伟；

【导师】 邓志锋；

【作者基本信息】 南昌大学 ， 外科学， 2010， 硕士

【摘要】 研究背景和目的:中枢神经系统(central nervous system,CNS)损伤修复一直是困扰神经科学工作者的重大难题。传统观念认为中枢神经系统一旦损伤便不能恢复,但随着近年来神经科学的发展,尤其是干细胞相关研究的不断深入,人们逐渐认识到成年CNS再生是由神经元的固有特性(intrinsic properties)和其所处的环境(environmental cues)共同决定的。因此增加受损神经元的再生潜能并改善轴突再生的微环境便成为CNS损伤修复研究的新的楔入点。神经损伤后修复的难点之一是神经元再生困难,随着近年来干细胞研究的迅猛发展,组织和细胞移植治疗成为学者们最为关注的方法之一,而选择合适的种子细胞则是细胞移植治疗的核心问题。目前用于移植的种子细胞供体主要有胚胎干细胞、神经干细胞(neural stem cells,NSC),以及其他具有成神经分化潜能的成体干细胞。尽管这些细胞在CNS损伤修复移植治疗中均显示出不同程度的积极作用,但由于存在细胞来源局限、取材不易、免疫排斥以及伦理道德等诸多方面的问题,临床应用受到很大限制。因此,寻找新的种子细胞来源成为当务之急。皮肤是人体最大的器官,包括来源于三个胚层的多种不同的组织细胞,自我更新和再生修复速度快,是成体干细胞分离和移植研究的良好对象。真皮多能干细胞(skin-derived precursors,SKPs)是一种新近分离成功的具有高度增殖能力和多向分化潜能的间充质干细胞,具有分化为脂肪细胞、肌细胞及神经细胞的潜能。相对于其他移植供体细胞而言,SKPs具有来源丰富、容易获得、可自体移植和具有更少的“谱系倾向性”等优点,又避免了胚胎伦理学、来源不足和异体排斥等弊端。相关研究表明,SKPs移植治疗脊髓损伤时,不仅能外在补充缺失神经元,同时可通过分化为Schwann细胞促进轴突再生及髓鞘化,显示了SKPs在CNS损伤修复中的良好应用前景。研究内容和方法:为了解SKPs移植对脑缺血大鼠中枢神经损伤的修复功能及神经保护作用,本实验进行了下列相关研究。1.利用悬浮成球及单层贴壁相结合的细胞培养方法,分离成人包皮组织,获得真皮来源细胞,无血清条件下添加B27及生长因子bFGF和EGF使其悬浮生长,培养数日后去除B27及生长因子,添加血清使细胞贴壁生长并快速扩增。获得SKPs细胞后,应用免疫化学方法检测其表面标志物CD29、CD34和Nestin的表达。2.线栓法制作大鼠大脑中动脉缺血再灌注损伤模型,CM-DiI标记SKPs后,通过股静脉移植到大鼠体内。通过抓握实验、平衡木行走实验及水迷宫实验对实验大鼠进行移植前后神经功能评分。3.免疫荧光法检测各组实验大鼠神经细胞(NSE、GFAP)相关抗原的表达,观察移植SKPs在实验大鼠体内存活及向神经细胞分化情况。4.利用TUNEL法分别检测各个时间点缺血对照组及移植组脑内神经细胞凋亡情况,镜下观察脑组织健患侧神经细胞凋亡数目。结果:1.采用悬浮成球方法分离培养人真皮来源SKPs,此时细胞扩增较慢,细胞贴壁生长后,细胞扩增速度加快,2-3d传代一次。根据SKPs特异性标志,对其进行免疫染色鉴定,体外单层贴壁培养的真皮多能干细胞,CD29和Nestin呈阳性表达,CD34阴性表达。2.通过抓握实验、平衡木行走实验及水迷宫实验在SKPs移植后1、3、5、7、14天五个时间点对正常组、脑缺血对照组及SKPs移植组大鼠进行神经功能评分。与正常组比较,缺血后1-14天,模型组各项神经功能检测指标均明显降低(P<0.05);观察期内有自发恢复特点,但仍低于正常组水平:与对照组比较,缺血后3、5、7、14d,SKPs移植组能够增加大鼠的抓握力(P<0.05);促进脑缺血大鼠平衡木行走的能力(P<0.05);与对照组比较缺血后5、7、14d能够明显促进脑缺血大鼠记忆功能的恢复(P<0.05)。3. SKPs移植1天可见移植的SKPs主要分布在脑组织缺血侧,表明SKPs经股静脉移植后可在短时间内迁移到脑缺血区域,由于我们在免疫组化中应用的是小鼠抗人的GFAP单克隆抗体和兔抗人NSE单克隆抗体,这些表达GFAP和NSE的细胞同时表达CM-DiⅠ,因此GFAP和NSE阳性细胞来源于移植的SKPs,表明SKPs经移植进入实验大鼠体内仍保留向神经细胞分化的特性。为进一步增加脑组织中外源性神经干细胞数量提供了一条思路。4.正常组和缺血对照组、SKPs移植组的健侧脑组织TUNEL染色只有零星的凋亡细胞。缺血对照组整个缺血区内均可见到凋亡细胞,大量成簇出现的凋亡细胞主要分布在梗死灶边缘区(皮层)的内侧。SKPs移植组凋亡细胞分布与缺血对照组相同,但皮层缺血半暗带区凋亡细胞数(57±15)与缺血对照组(102±7)比较明显减少(P<0.05)。结论:我们利用悬浮成球及单层贴壁相结合的培养方法成功地从人包皮组织中分离并扩增了SKPs。移植SKPs到脑缺血损伤大鼠体内能够存活并定向迁移至脑缺血部位并向神经细胞分化,同时可减少缺血区的神经细胞凋亡,促进大鼠脑缺血损伤后的神经功能恢复。结果提示移植的SKPs可能是通过向神经细胞的分化、抑制缺血区神经细胞的凋亡来促进缺血损伤后的神经再生和功能重建,SKPs有望成为干细胞移植治疗的新的种子细胞来源。更多还原

【Abstract】 The recovery of central nervous system(CNS)after injury is all along a difficult problem that puzzles the neuroscientists.In the past,it was widely accepted that once CNS was injured,nothing could be done.Since recent two decades,numerous studies have been carried out by neuroscientists and it is concluded that CNS after injury can be recovered.The course of recovery based on two factors:intrinsic properties and environmental cues.So,it is aimed to promote the regeneration of neurons and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.In regenerative medicine,some encourage results have been obtained.It is well known that cell replacement therapies show particular promise in the nervous system,and transplanted embryonic stem cells(ESCs)or neural stem cells (NSCs) have been shown to promote functional recovery in animal models,for example,spinal cord injury (SCI).Many stem cells (such as ESCs and NSCs) can be used to treat a lot of neurological diseases. Although the therapeutic potential of such transplants is clear,a number of problems, such as insufficient cell number, ethical issues, immunosup- pression, and so on,limit their application in clinic.In this background,a new type of stem cells called skin-derived precursors (SKPs) was discovered in adult mammalian skin.They can differentiate into cells of both neuroectodermal and mesodermal lineage,including(perhaps not limited to) neurons,glia,osteogenic cells and adipocytes.Compared with the above cells, SKPs, which own multi-oriented differentiative potentialand self-renewal capability,have an accessible,potentially autologous tissue source and can expand in vitro cell culture.So they have important therapeutic implications.In order to determin if the SKPs have the effects of promoting the regeneration and function recovery of Ischemic brain injury. a series of studies were carried out as follows: (1) Combination of suspension and adherent culture method , digested adult dermal tissue, Serum-free conditions, Add B27 and growth factors bFGF and EGF to suspended growth. Immunochemistry to detect the surface markers CD29, CD34 and Nestin. (2) Middle cerebral artery occlusion (MCAO) models were produced by obstructing MCA using a single strand nylon thread, CM-DiI labeled cells, Through the femoral vein transplanted into rats. Neurological function was evaluated with beam-walking,bar-grasping and water maze. (3) Immunofluorescence assay (NSE, GFAP)-associated antigen expression of rat nerve cells. To investigate the survival and differentiation of skin-derived precursors transplanted into rat brain.(4) TUNEL kits were used to detect the control group and transplant brain tissue neural cell apoptosis. Microscopic observation of brain tissue in the number of neuronal apoptosis.Main results are as following:1. Combination of suspension and adherent culture , According to SKPs specific signs, Most cells expressed CD29 and Nestin instead of CD34b y immunofluorescent staining.2. ischemia group rats and SKPs transplantation group rats showed severe neurological disfuction compared with sham-operated group(p<0.05). Rats receiving SKPs had a more rapid recovery of beam-walking,bar-grasping and water maze performance than ischemia group rats(p<0.05).3. Twenty-four hours later, the transplanted SKPs can be detected mainly in the ischemic zone. These suggested that the transplanted SKPs can migrate to the ischemia zone through the femoral vein graft in a short time. The immunofluorescent staining results confirmed a part of NSE and GFAP positive cells expressed CM-DiI also. These data suggested that the number of nerve cells differentiated from SKPs. In order to further increase the number of exogenous neural stem cells provides a train of thought.4. TUNEL: After 2 hours of MCA occlusion and 24 hours reperfusion, apoptotic cells exhibiting DNA fragmentation increased in the regions of caudoputaminal and cortex, groups of apoptotic cells were primarily localized to the inner boundary zone of the infarct. The number of apoptotic cells of SKPs transplantation group(57±15)decreased significantly compared with ischemia group(102±7)in the penumbra of cortex with light microscopic analysis (x400 field),p<0.05.Conclusion: Combination of suspension and adherent culture , Skin-derived precursors (SKPs) were isolated from the adult dermal tissue,cultured and identified by their multilineage differentiation capacity. SKPs can survive in the brain of MCAO rats and were mainly distributed in the region around the ischemic focus and remained the differentiation characters of neuron cells. The results demonstrate that SKPs can improve the neurological function follwing focal cerebral ischemia in rats. Manifested as behavioral score was significantly higher、to reduce the number of neuronal apoptosis in Damage zone. The results suggest that transplant SKPs may be through to Differentiat into neural cells and inhibit neuronal apoptosis in ischemic area to promote nerve regeneration and functional reconstruction after ischemic injury,For clinical treatment of ischemic brain injury provides a new way.更多还原