【作者】 徐启飞；

【导师】 周初松；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 硕士

【摘要】 脊髓损伤(Spinal cord injury,SCI)的治疗一直是困扰全人类的一个难题,尤其是伴随着现代社会的快速发展和人们生活节奏的日益加快,各种创伤导致的脊髓损伤的发生率逐年攀升。据统计我国每年有脊髓损伤病人约5万人,且以每年10%的速度不断上升。由于脊髓损伤较高的致残率,且大多数患者需要长期住院治疗和康复,给家庭社会带来了沉重的负担。人们尝试过无数治疗脊髓损伤的方法,但都不大理想。近年来,随着神经病理生理及神经发育学研究的不断深入,神经组织不能再生的传统观念受到了挑战。尤其是神经干细胞(Neural stem cells,NSCs)的发现和证实,为治疗包括脊髓损伤在内的难治性中枢神经系统(Center neural system,CNS)疾病开辟了新的道路。神经干细胞具有分化为神经元、星型胶质细胞、少突胶质细胞的能力,并具有很强的自我更新能力和增殖潜能,因此具有非常重要的理论研究和临床应用价值。通过移植大量的细胞特别是神经干细胞来促进损伤脊髓的功能恢复已经取得了令人鼓舞的结果,但诱导动员内源性神经干细胞来修复脊髓损伤方面的研究关注较少;与移植细胞相比,激活内源性神经干细胞因是利用自身资源,它不仅克服了从组织获取神经干细胞的危险性和局限性,也避免了组织细胞移植中存在的伦理、免疫排斥、来源有限等问题,因此在治疗中枢神经系统损伤和神经退行性疾病领域将会有广阔的应用前景。虽然神经干细胞的研究取得了一定的进展,但对内源性神经干细胞仍了解很少,尤其是对其体内的调控机制仍不明确。因此,在本研究中,利用BrdU/Nestin标记内源性神经干细胞的方法,我们观察了大鼠脊髓损伤后内源性神经干细胞增殖过程及Wnt-1表达的规律,探讨了大鼠脊髓损伤后内源性神经干细胞自我增殖变化规律及其可能机制。此外,鉴于Wnt/β—Catenin信号通路在中枢神经系统的发育和神经干细胞的增殖分化中起了重要作用,我们探索了是否应用氯化锂能提高脊髓损伤后内源性神经干细胞的增殖。第一部分大鼠脊髓损伤后内源性神经干细胞增殖过程中Wnt-1的表达目的:探讨大鼠脊髓损伤后信号蛋白Wnt-1的表达及其与脊髓内源性神经干细胞(ENSCs)早期增殖的关系。材料及方法:(一)雌性SD大鼠50只,随机均分为正常对照组(A组)、损伤组(B组)。(二)A组仅暴露椎板,B组采用经典的Allen法建立脊髓损伤模型。(三)于损伤后1天、3天、1周、2周、4周进行取材,对距离损伤中心5mm的脊髓行免疫组化染色,检测ENSCs的增殖及Wnt-1蛋白表达的动态变化,统计分析二者之间的相关性。结果:A组:可见脊髓外周及室管膜区域极少数细胞呈BrdU/Nestin阳性,排列稀疏,灰白质中几乎无BrdU/Nestin阳性细胞。与A组相比较,B组BrdU/Nestin阳性细胞在损伤后1d明显减少,3d后开始增加,并超出A组水平,主要见于损伤脊髓组织周围及室管膜区,灰质和白质中出现散在点片状和丝状表达,7d时BrdU/Nestin阳性细胞数达到高峰。BrdU/Nestin阳性细胞主要见于室管膜以及环绕脊髓的软膜,并向脊髓中央延伸。14d时BrdU/Nestin阳性细胞开始下降,28d后明显下降。A组中仅见极少量Nestin的表达。B组在损伤后1d可见少量Nestin表达,3d时开始增加,7d时可见较多Nestin阳性面积的表达,主要分布于室管膜周围区域及灰质中,14d时开始逐渐减少,28d后Nestin阳性细胞明显减少。Nestin表达阳性的树状突起延伸至灰质,在灰质中的表达主要分布于神经元附近和血管周围,个别细胞形态类似星形胶质细胞,同时在灰质偶见少量类似神经元样Nestin阳性细胞。A组脊髓组织内Wnt-1无明显表达。与A组相比,B组各时间点均可见Wnt-1的表达。1d时就可见脊髓中央管周围及外膜Wnt-1表达阳性区域的出现,脊髓灰白质内少量表达,较为分散,3d后阳性区域的面积达高峰,7d时维持在较高水平,之后逐渐减少,28d后仅见极少量Wnt-1阳性表达。结论:正常大鼠脊髓中存在着有增殖能力的内源性神经干细胞,它们保持增生活性状态,在损伤后明显被激活,从而快速增殖,这一过程发生的机制可能与Wnt信号通路有关。第二部分:氯化锂对大鼠脊髓损伤后内源性神经干细胞的影响目的:探讨氯化锂对大鼠脊髓损伤后内源性神经干细胞的影响。方法:75只成年雌性SD大鼠,随机分为正常对照组(A组)、单纯损伤组(B组)、损伤后氯化锂治疗组(C组)。A组仅行椎板暴露,B组和C组采用经典的Allens法在T10段制作急性脊髓损伤模型,C组于损伤后1小时给予氯化锂治疗,每日3mmol/Kg腹腔注射,直至取材;B组以相同的方法给予等量生理盐水。各组分别于损伤后1d、3d、1周、2周、4周进行Brdu腹腔注射标记,共3次,最后一次标记后4小时取材,对距离损伤中心5mm处的脊髓进行Brdu、β—Catenin的免疫组化检测,图像分析软件进行Brdu阳性细胞计数及β—Catenin阳性表达面积的计算。结果:A组脊髓中央管周围及外膜可见少量Brdu阳性细胞,几乎无β—Catenin阳性表达。B组脊髓损伤后24小时即可见多量Brdu阳性细胞及微量β—Catenin的表达,1周时两者达到高峰,主要见于损伤脊髓周围的灰质和室管膜区,2周后明显开始减少,4周时仅可见少量Brdu阳性细胞及β—Catenin的阳性表达。C组Brdu阳性细胞及β—Catenin的表达在脊髓损伤后24小时与B组无显著性差异,1周时在中央管周围、外膜、灰质中Brdu阳性细胞数及β—Catenin的表达量明显较B组多,至2周时仍处于较高水平,4周时仍有多量Brdu阳性细胞及β—Catenin的表达。结论:氯化锂在大鼠脊髓损伤后能促进内源性神经干细胞的增殖,其机制与Wnt信号通路有关。小结:1.正常大鼠脊髓中存在内源性的神经干细胞,他们处于“静止”或“休眠”状态,在损伤等特定因素的作用下其分裂、分化的潜能被激活,从而出现增殖现象。2.脊髓损伤后,伴随着内源性神经干细胞增殖能力的逐渐增强,Wnt-1的表达也逐步增多,这提示Wnt-1,或者进一步说Wnt信号通路可能参与了脊髓损伤后内源性神经干细胞的增值过程。3.应用氯化锂可促进大鼠脊髓内源性神经干细胞的增殖,说明氯化锂可能在脊髓损伤的治疗中发挥积极的作用。更多还原

【Abstract】 The treatment of Spinal cord injury (SCI) has always been a big difficulty to all of humanity in a long period of time. Especially with the rapid development of modern society and the ever-accelerating pace of life, the incidence of all kinds of traumatic SCI is increasing year by year. According to the statistics, there is about 50,000 SCI patients each year in our country, which is increasing on a rate of 10%. SCI brings a heavy burden to family and society because of high rate of deformity, long-term hospitalization and rehabilitation. People has tried numerous methods to treat it, but no one is ideal. On recent, following development of study of neuro-pathophysiology and auxology, the conventional idea that nervous tissue do not regenerative had challenged. The discover and confirm of Neural stem cells have brought a new way for intractable diseases of center neural system including SCI.Neural stem cells(NSCs) are a kind of cells that have the ability to differentiate into neurons, astrocytes and oligodendrocytes. They have self-renewal activity and multi-potential proliferation, which is important for theory research and clinical applications. The transplantation of a variety of cells, particularly the neural stem cells or NPCs, in multiple central nervous system (CNS) injury paradigms has provided to a certain degree encouraging results for functional recovery. While induction and recruitment of endogenous adult NSCs, has received relatively little attention. Compared with transplantation of cells,the activation of endogenous NSCs is use self-resource. It not only get rid of the limit and danger of acquiring NSCs from tissue, but also avoid the exiting questions of ethics , immunological and limited source. It will have a promising future in the treatment of head trauma and degenerative diseases of the central nervous system.Although we have got some progress on neural stem cells, little is knowen about the proliferation, migration, and differentiation of adult NSCs in the mammalian spinal cord, especially the mechanism of regulation in vivo. So used bromodeoxyuridine(BrdU)/Nestin to label the endogenous NSCs in this study, we study the proliferation and differentiation of endogenous neural stem and the expression of Wnt-1 after spinal cord injury in rat for the aim to explore the self-duplication regularity of endogenous NSCs. In addition, Wnt/β—Catenin signaling pathway has been recently shown to be an important role in the development of central nervous system and in vivo proliferation of NSCs. Therefore, we examined whether exogenous administration of Lithium chloride(LiCl ) would further increase the numbers of NSCs after SCI in vivo.Part I : Experimental study on the expression of wnt-1 in the proliferation process of endogenous neural stem cells after spinalcord injury in ratObjective: To investigate the Expression of wnt-1 in the earlier proliferationprocess of endogenous neural stem after spinal cord injury(SCI) in rats.Methods:(1)Fifty Wister rats were randomly divided into two groups: the normal control group (group A) and the spinal cord injury group (group B). (2) A group only exposed the vertebral plate, B and C group SCI models were established by Allen method.(3) The rats were killed at 1d, 3d, 1,2 and 4 weeks after injury, the expression of wnt-1 and the proliferation of ENSCs were analyzed in the samples which were got from the two levels of the spinal cord with the length of 5mm harvested both rostral and caudal region to the injury center by immunoreactivity. The linear regression analysis was used to evaluate the correlation between the expression of wnt-1 and the proliferation of ENSCs.Results: Group A shows that there is a very small number of Brdu/Nestin positive cells in the peripheral and ependymal region of spinal cord , and with sparse in the gray matter and white matter. Twenty four hours after SCI there is visible portion of Brdu/Nestin positive cells,seen mainly in the surrounding of injured spinal cord and ependymal areas in Group B, which appeared in scattered spots flake and filament expression in the gray matter and white matter. It begins increasing in three days, and reaching the peak in one week after SCI. BrdU / Nestin-positive cells were seen mainly in ependymal and meninx vasculosa around the spinal cord, and extent to the centre of the spinal cord. The number of Brdu/Nestin positive cells Starts to decline two weeks, and show up significant decrease in four weeks after SCI.Almost no expression of Nestin exists in group A. One days after SCI, a small number of Nestin expression begin showing up, which can be seen more in seven days after SCI, mainly distribute in the ependyma area and gray matter. It gets the beginning of decline gradually in two weeks after SCI, and decreases significantly after four weeks. The dendrite with expression of Nestin extend to the gray matter, the expression in gray matter is mainly distributed in neurons and blood vessels. Some cells have the similar morphology with astrocytes. Occasionally a small amount of neuron-like Nestin-positive cells appear in the gray matter. There is no obvious Wnt-1 expression in Group A, and Group B haves the contradictory phenomenon. one day after SCI expression of Wnt-1-positive region can be seen around the central canal and adventitia of spinal cord, little appears in the white and gray matter,which is quite dispersed. In three days the expression reach a peak, and stay at a relatively high level till one week after SCI, which followed by a gradual decreasing process, in four weeks only a very small number of positive expression of Wnt-1 can be seen.Conclusions: Normal rat spinal cord has the presence of endogenous neural stem cells, which own proliferative capacity and maintain a state of proliferative activity. These cells can be obviously activated by the stimulation of injury through one mechanism that may be associated with Wnt signaling pathway.PartⅡ: Effect of Lithium chloride on endogenous neural stem cellsafter spinal cord injury in ratsObjective: To investigate the effect of Lithium chloride on endogenous stem cells after spinal cord injury in rats.Methods: Seventy five female Wister rats were divided into three groups randomly: the control group (group A); spinal cord injury group(group B); lithium group(groupC). A group only exposed the vertebral plate, B and C group were made a injury at T10 level by Aliens method. The rats in C group were injected with lithium 3mmol/Kg every day after SCI, while the rats in B group received same amount of 0.9% normal saline by the same way. All rats were labeled three times with Brdu at 1d, 3d, 7d, 14d and 28d. The equivalent rats were killed four hours after the last label in all group. The spinal cord with the length of 5mm to the injury center were the samples were analyzed by Brdu and p—Catenin immunohistochemical detection to study the response of endogenous stem cells.Results: There was few Brdu positive cells and less expression of p—Catenin in the center canal and adventitia of spinal cord in group A. many Brdu positive cells and little expression ofβ—Catenin were found in gray matter and ependyma of injury area in group B 1d after SCI, which reached the peak at 7d, and declined gradually 14d postinjury. Just a few Brdu positive cells and little expression ofβ—Catenin existed 28d after SCI. There was no difference between B and C group on the Brdu positive cells and the expression ofβ—Catenin 1d after SCI. Group C had more Brdu positive cells and the expression ofβ—Catenin in the center canal and adventitia of spinal cord, which were also at the high level 14d and 28d after SCI. Conclusions: Lithium could activate the endogenous stem cells after SCI, the mechanism may be relate with the Wnt signal pathway.Conclusion:1. Endogenous neural stem cells exist in normal rat spinal cord, which are staying in "static"or "hibernation" state. In the role of specific factors such as injury, their potential of division and differentiation is activated, and thus give rise to proliferation.2. After SCI the capacity of endogenous neural stem cells increase by degrees, which is accompanied by the gradual mult of expression of Wnt-1, all of this prompt that Wnt-1 or Wnt signaling pathway may be involved in proliferation process of endogenous neural stem cells after SCI.3. The application of lithium chloride can promote proliferation of endogenous neural stem cells in rat, which reveals that lithium chloride may be playing an active role in the treatment of SCI.更多还原