【作者】 翟翠静；

【导师】 曹友德；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 第一部分新生大鼠神经干细胞的分离、培养研究背景与目的神经干细胞(neural stem cells, NSCs)是近年来神经科学领域的研究热点。NSCs是具有自我更新能力和多向分化潜能的细胞,可分化为神经系统的神经元、星形胶质细胞和少突胶质细胞,在胚胎及成年哺乳类动物中广泛存在。NSCs的分离培养是其基础和应用研究的前提,本部分目的在于获得新生大鼠NSCs,为深入研究其增殖分化及在SCI中的应用奠定基础。方法从新生24h内大鼠端脑及中脑部位分离NSCs,采用体外无血清悬浮培养,以获得能在体外长期生存的NSCs;通过免疫细胞化学检测NSCs标记物nestin表达及分化为神经元和胶质细胞的能力。结果获得的NSCs能在体外长期生存,具有很强的增殖能力、自我更新能力,表达nestin,具有多向分化能力,能分化为神经元和胶质细胞。结论成功地从新生大鼠端脑和中脑中分离得到了能在体外长期培养的NSCs,培养的NSCs表达nestin,有自我更新、多向分化的特点,通过培养细胞大量扩增。第二部分低强度超声联合神经干细胞移植对大鼠脊髓损伤功能恢复的实验研究研究背景及目的脊髓损伤(spinal cord injury, SCI)的修复一直以来是神经科学研究领域的一大难题,由于脊髓组织自身的修复能力极其有限,激活的内源性神经干细胞再生不足以修复损伤的脊髓组织,因此,利用外源性细胞移植来替代丢失的神经细胞修复受损的脊髓、重建神经通路一直以来是人们研究的热点。神经干细胞因其来源于神经组织、具有自我更新能力、具有分化为神经元和神经胶质细胞的潜能、能迁移到达损伤或疾病部位等特点,成为脊髓损伤细胞替代疗法的理想移植材料。近年来,随着对低强度超声生物学效应的研究发现,低强度超声具有促进细胞生长、帮助实验性周围神经损伤的一定作用。本实验旨在观察源于中脑端脑的神经干细胞移植联合应用超声对脊髓损伤功能恢复的作用。方法40只成年SD大鼠随机分为正常A组对照组、B组损伤组(SCI组)、C组单纯移植NSCs组(NSCs)和D组NSCs移植联合应用超声组(NSCs+ LIU ),每组10只。除A组外,其余3组制作T8平面脊髓全横断模型,将含有NSCs悬液的明胶海绵植入C组和D组横断损伤脊髓处,B组在损伤处移植含细胞培养液的明胶海绵,D组同时应用超声治疗。2月后,实验各组采用BBB评分、皮层体感诱发电位(CSEP)、电镜和免疫组织化学观察大鼠运动传导功能恢复的作用。结果对照组BBB评分21分。术后2月BBB评分,B组(2.5±1.05)分,C组(5.0±0.89)分,D组(6.8±0.75)分。C、D组评分明显高于B组,以D组最高(P<0.05)。但未达到正常。脊髓损伤后,B、C、D组的CSEP波消失,术后2个月C组D组的波形有所恢复,但潜伏期延长,波幅幅度降低,而B组波形仍完全消失。电镜显示,C组、D组损伤区见再生髓鞘、轴突及神经元。而B组仍见明显的髓鞘细胞变性及瘢痕组织。免疫组织化学显示D组移植的NSCs分化为神经元数量较多,分化为胶质细胞的数量较少,且形成的神经元形态较规则,神经纤维走行较规则。结论NSCs移植联合应用低强度超声能促进损伤脊髓运动和传导功能的部分恢复。更多还原

【Abstract】 PART ONE ISOLATION AND CULTIVATION OF NEONATAL RAT NEURAL STEM CELLSBackground and Objective Neural stem cell, the self-renewing multipotential cell, could generate neural tissue, including neurons, astrocytes and oligodendrocytes. It existed not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including human. Isolation and culture of NSC was the basement of the fundamental and applied research. Objective of this part was to obtain neonatal rat NSCs for further study on its proliferation and differentiation.Methods Isolation the primary NSCs from endbrain and midbrain of neonatal rats(in 24h after birth), the cells were suspended and cultured in serum free medium (containing bFGF and EGF ) for long-term survival in vitro. Technique of single-cell clone culture was used to confirm the character of self-renewing. Ability of multi-directional differentiation and specific antigen, nestin, were detected by immunocytochemistry. Cytometry was used to evaluate the proliferation capability of NSCs and the growth curve is plotted.Results NSCs could survive in vitro for long time. They showed multipotential of differentiate into neurons and glial cells and the ability of self-renewing and proliferating. The NSCs were positive for nestin expression.Conclusions The NSCs were isolated from endbrain and midbrain of neonatal rats. These cells could be cultured for long time in vitro. They expressed nestin and had the ability of self-renewing and multi-directional differentiation. Cells could expand in serum free medium plus mitogen factors.PART TWO FUNCTIONAL RECOVERY OF RATS WITH SPINAL CORD INJURY TREATED BY NEURAL STEM CELL TRANSPLANTATION COMBINED WITH LOW INTENSITY ULTRASOUNDBackground and Objective Recovery of spinal cord injury(SCI) was a great puzzle in the neuroscience research domain for a long time. At present neural stem cells(NSCs) were utilized to treat SCI and low intensity ultrasound(LIU) could improve the experimental growth of peripheral nerves. Objective of this part was to observe the function recovery of rats with spinal cord injury(SCI) transplanted by neural stem cells(NSCs) and combined with low intensity ultrasound(LIU) application .Methods Forty SD rats were randomly divided into four groups. Group A: control group,Group B: spinal cord injury group (SCI), Group C:spinal cord injury with NSCs transplantation(NSCs), Group D:NSCs transplantation combinded with low intensity ultrasound(NSCs+LIU) application, each group had 10 rats. The rats in the Group B、C and D were made into the models of T8 plane with full transection of spinal cord ;the gelatin sponge with the cultured NSCs suspension of rats was grafted into the injured spinal cord in the group C and D while the gelatin sponge with the equal volume cell culture solution was transplanted into the rats in the group B. The rats in the Group D were utilized ultrasonic therapy meanwhile. Two months after operation, the motor and conduction function of spinal cord was determined by using BBB scores, cortical somatosensory evoked potential (CSEP), electron microscope and immunohistochemistry.Results As assessed by BBB scores, the rats in the Group B、C and D recovered but not recovered to the normal leve1, the Group D was the best after operation, CSEP of the Group B was still disappeared. Meanwhile in group C and D, the CSEP wave appeared again,but the waveform of the Group C and D was prolonged and the amplitude of wave was degraded slightly.Axons and myelinsheath regrown in group C and D, and nerve cells were discovered in group D cheked by electronic microscope. The quantities of neurons of NSCs differentiation were more and their appearances were more regular by immunohistochemistry in the Group D.Conclusions NSCs transplantation combined with low intensity ultrasound can improve the partial recovery of motor and conduction function of injured spinal cord in rats.更多还原