【作者】 巴迎春；

【导师】 王廷华； 李力燕；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【摘要】 [目的]：探讨神经干细胞(NSC)脊髓内移植对脊髓全横断(SCT)损伤大鼠运动功能恢复的影响,并研究其与神经再生,神经营养因子(NTF)表达的关系,为应用NSC移植治疗SCI提供理论依据。[方法]：正常成年SD雌性大鼠85只,分为两批；第一批15只动物用于BDA条件实验,分为双侧大脑皮质注射10% BDA 0.5μL／位点,存活2w组；单侧大脑皮质注射10%BDA 1 p L/位点,存活3w组；双侧大脑皮质注射10%BDA 0.5μL/位点,存活3w组。每组各5只动物。第二批SD大鼠共70只,分为3组：①假手术组,②单纯全横断组；③神经干细胞移植组；分别对每组每只动物进行BBB评分、诱发电位CSEP和MEP、电针刺激大脑皮质运动区的测定,观察行为学,神经电生理学变化；对各组动物分别进行BDA追踪,免疫组化染色,RT-PCR检测,观察NSC移植效果,植入细胞存活,分化,宿主神经再生,神经营养因子表达,信号分子变化,以探讨NSC治疗脊髓损伤的分子机制。[结果]：1.在3组进行BDA追踪实验的大鼠中,双侧大脑皮质注射10% BDA 0.5μL/位点,存活3w组的追踪效果最好。2.通过行为学,神经电生理等指标检测,确定大鼠脊髓全横断损伤动物模型构建成功；且进一步证实脊髓神经具有可塑性。3.NSC移植后大鼠的后肢功能BBB评分优于单纯脊髓全横断组。NSC移植后大鼠的皮层体感诱发电位(CSEP)、运动诱发电位(MEP)的潜伏期短于单纯脊髓全横断大鼠。电针刺激大脑皮质运动区发现NSC移植组大鼠双后肢出现明显收缩,且以对侧更为明显。说明NSC移植能促进大鼠感觉,运动行为与神经电生理功能的部分恢复。4.BDA神经束路追踪和5-HT、CGRP免疫组化染色结果显示,损伤脊髓头侧1-3节段的纤维数量明显减少,尤其是临近疤痕的节段。但随时间进程,脊髓存在有限的神经再生。而NSC移植能明显加强这种神经再生(与单纯脊髓全横断组比较)。5.NSC移植明显下调损伤位点头侧脊髓神经生长因子TGF-betal、CNTF和促凋亡基因Bax、CASPESE-3以及相关信号分子BAD的表达；同时明显上调凋亡抑制基因BCL-2的表达；但不改变细胞周期蛋白调控基因CDK4和CyclinD1、CyclinE和BDNF的表达。[结论]：1.BDA条件实验说明,本实验中所使用的BDA示踪剂(美国Molecular Probes公司)是有效的,在双侧大脑皮质上选择多点注射,以每个位点注射0.5μL的10%BDA,注射后大鼠存活3w为最佳的BDA追踪实验方案。2.脊髓全横断模型是成功的,所使用的手术方法是可行,有效的；此外,脊髓损伤后随时间延长,CST纤维有少量再生,说明脊髓具有神经可塑性。3.NSC移植能促进宿主感觉及运动传导功能的部分恢复。4.NSC移植可能通过影响神经再生、细胞凋亡,和调节NTF及信号转导通路分子的表达水平,来实现促进脊髓修复的目的。更多还原

【Abstract】 [Objective]:To explore the effect of neural stem cells transplantation on motor function recovery in injuried spinal cord, then investigate the relation among NSC and nerve regeneration, expression of neurotrophic factor, so as to provide the possibility by using neural stem cells transplantation.【Method】:Adult SD female 85 rats were divided into two parts; the first part consisted of 15 SD rats were performeded BDA condition experiment,the rats were divided into three groups,each group consisted of 5 rats:the first group rats survived for 2w after 10%BDA was injected into both cerebral cortex, 0.5μL/site. while 10%BDA was injected into single cerebral cortex, 1μL/site, in the second group rats; in the third group rats,10%BDA was injected into both cerebral cortex,0.5μL/site, the rats survived for3w.The second parts consisted of 70 SD rats were examined the effect of neural stem cells transplantation,the rats were divided into three groups:①sham operation,②merely spinal cord transsection group,③NSC transplantation group; After NSC transplantation into spinal cord,BBB score, evoked potential(CSEPand MEP), cortical motor area stimulation by electro-acupuncture, BDA tracing, immunohistochemistry staining and RT-PCR were performed to determined the cell survival, differentiation, host nerve regeneration,the expression of neurotrophic factor, as well as the change of signaling molecule. [Result]:1. The BDA trace effect of the third group rats that survived for 3w after 10%BDA injection into both cerebral cortex is best one(0.5μL/site).2. spinal cord transection animal model is reliable by the experiment of neuroethology and neurophysiology; neuroplasticity has occurred,following injury in the spinal cord.3. BBB score of hind limb function in neural stem cells transplantation rats is better than merely spinal cord transaction group. Latency of CSEP and MEP is shorter after neural stem cells transplantation. Neural stem cells transplantation leads to hind limbs contraction after cortical motor area stimulation by electro-acupuncture,especially in opposite side.Moreover,neural stem cells transplantation can promote partial recovery of rats sensation, motor behavior and neurophysiology.4.Nerve fibers quantity by BDA tracing,immunohistochemistry staining of 5-HT,CGRP, showed that SCI results in the fibers quantity obviously decreased,especially in segment closed to the scar.moreover, limited nerve regeneration occurred with time course in spinal cord,and neural stem cells transplantation can obviously promote the regeneration(compared with merely spinal cord transsection).5. Neural stem cells transplantation also can obviously down regulate the expression of Bax, CASPESE-3 genes,and TGF-betal and CNTF as well as BAD as signaling molecule which promoted Apoptosis, Whereas,it can obviously up-regulate the expression of BCL-2 as gene which can inhibit Apoptosis,but it can not change the expression of controlling gene of cyclin including CDK4 and CyclinDl,CyclinE, BDNF. [conclusion]:1. BDA condition experiment demonstrated that BDA tracing is effective in the experiment rats. Multitude site injection should be chosen in both cerebral cortex. Rats survived for 3w after 10%BDA was injected into both cerebral cortex is the best BDA trace proposal (0.5μL/site)among the three traced groups.2. Spinal cord complete transection was successful, operation method was feasible and reliable; it showed plasticity after injury.3. Neural stem cells transplantation can promote functional recovery,and neuroplasticity.4. Neural stem cells transplantation promote the recovery of injuried spinal cord, the expression of neurotrophic factor genes, apoptosis genes and signal transduction molecules.更多还原