【作者】 肖明；

【导师】 胡刚； Fred J.Roisen；

【作者基本信息】 南京医科大学 ， 药理学， 2005， 博士

【摘要】 脊髓损伤修复是目前神经科学研究领域最热门的课题之一。过去十几年来,有关脊髓损伤与再生的实验研究取得了很大的进展,出现了许多新的实验治疗方法与手段,不同程度地促进了动物模型的脊髓修复与功能恢复。但在将这些治疗措施用于临床治疗之前,有关疗效与生物安全性等方面关键问题必须予以考虑或解决。因此,寻找一种既容易获取又具有良好的修复潜能的成人神经前体细胞,用于自体移植治疗脊髓损伤,显得具有特别重要的临床意义。 既往实验证实嗅神经上皮存有可替代神经元与支持细胞的内源性前体细胞(基底层细胞),终生具有自我修复能力。因便于取材,又具有良好的自我修复特性,嗅神经上皮可作为通过体外培养获取成人神经干细胞,用于自体细胞移植的独特来源。在过去几年内,我们建立了成人嗅神经上皮前体细胞(ONPs)鼻腔镜活检技术和培养流程。活检的鼻腔嗅上皮组织,经过原代培养,通常有神经球形成。对神经球细胞(NSFCs)进行传代培养与生物学鉴定,证实其具有神经前体细胞的特征,并表达与分泌包括脑源性神经生长因子(BDNF)在内的数种神经营养因子。本动物实验,我们选择了对外源性BDNF敏感的红核脊髓束(RST)为研究对象,通过颈髓部分半横断术制作亚急性大鼠脊髓损伤模型,观察成人ONPs移植对切断的RST修复作用。主要研究内容如下:1)ONPs移植入损伤脊髓后,更多还原

【Abstract】 Repair of Spinal cord injury is one of the most challenging issues in the present field of Neuroscience. In the past decade, basic science advances in spinal cord injury and regeneration research have led to a variety of novel experimental therapeutics designed to promote functionally effective axonal regrowth and sprouting. Before they are used to treat patients with spinal cord injuries, there are many issues to be considered relative to treatment safety and efficacy. A more readily accessible source of human cells, preferably with the potential for autologous transplantation, is needed.The olfactory neuroepithelium (ONe) undergoes life-long repair through the action of endogenous progenitors that replace its’ neuronal and supporting cells. The ONe, by virtue of its accessible location, has special advantages as a source of CNS progenitors for autologous transplantation. In the last several years, our laboratory has developed endoscopic biopsy and culture procedures for obtaining human adult ONe-derived progenitors (ONPs). These progenitors can be removed from the primary cultures following their formation of neurospheres.The neurosphere forming cells (NSFCs) have been characterized as neural progenitors and shown to produce neurotrophic factors including brain derived neurotrophic factor (BDNF). To evaluate the therapeutic potential of ONPs on spinal cord injury, the rubrospinal pathway was selected because it has been shown to be BDNF dependent. Following partial cervical hemisection, an axotomized rubrospinal tract (RST) model of subacute spinal cord injury was used to determine if, 1) engrafted human adult NSFCs survived and integrated into the damaged spinal cord; 2) rescued axotomized rubrospinal neurons (RSN) from retrograde atrophy; 3) promoted axotomized RST axonal regeneration, reinnervation and forelimb functional recovery. Our long-term goal is to develop procedures through which a victim of spinal cord injury or other neurological degenerative conditions could serve as the donor of progenitors for his/her own restorative grafts without the need for immunosuppression or ethical controversy.Characterization of GFP-labeled human adult ONPs in vitroThe two randomly selected NSFC lines were transfected with green fluorescent protein (GFP) using the LERS-EGFP vector; approximately 60% of viable cells 4 days after transfection were fluorescent. In Minimal Essential Meduium (MEM) plus 2.5% fetal bovine serum (FBS), the GFP-labeled NSFCs (GFP-NSFCs) consisted of heterogeneous populations in which some cells exhibited immunoreactivity to nestin (~40%), neural cell adhesion molecule (NCAM, ~30%), β-tubulin Ⅲ (NST, ~95%), A2B5 (~5%), Trk pan (~20%), Trk A (~7%) and Trk B (~13%). No immunoreactivity wasobserved for glial fibrillary acidic protein (GFAP) and Trk C. Immunoreactivity for BDNF was detected throughout the cytoplasm and on the surface of most NSFCs (~ 90%).Consistently, the expression of mRNA and protein for BDNF in the two GFP-labeled NSFCs lines was demonstrated by RT-PCR and Western Blot respectively. By RT-PCR, cDNA amplification products representative of BDNF (298 bp) mRNA were detectable in RNA prepared from the two NSFCs lines. The immunoreactive bands for BDNF (14 kDa) were also observed in lysates of both NSFCs lines.To determine the endogenous and secreted levels of BDNF in the two GFP-NSFCs, cell culture supernates and cell lysates ELISA were performed. The concentration of BDNF in supernates was equivalent for both NSFCs, was 220.28 ± 8.42, 224.51 ± 7.91 pg/106 cells/ 24 h respectively. The amount of BDNF in lysates of both NSFCs lines was 39.28 ±7.51, 38.57 ± 8.43 pg/mg protein respectively.Furthermore, the bioactivity of cell culture supernates from the two NSFCs was assayed based on neurite outgrowth from embryonic chick DRG neurons. E8 chick DRG explants grown for 48 h in purified BDNF or supernates from GFP-labeled NSFCs showed extensive process outgrowth.In summary, differentiation potential of the two GFP- NSFCs in the MEM plus 2.5% FBS was restricted along a neuronal lineage ha更多还原