脉络丛上皮细胞移植对脊髓损伤的修复作用及其机制

Spinal cord injury (SCI) is a disease that highly disables the suffers, and often results in high social and economic burden. Currently, there is no effective medical therapy available to restore the neural function in patients with SCI. In recent years, the transplantation of the choroid plexus has been tested for its potential in neuroprotection against central nervous system trauma in several animal models, for example, stroke and Huntington's disease. The role of the choroid plexus in growth and nutrient production makes it a novel candidate source of transplantation. Our research team has established a simple and practical way of primary cell culture, which prepares choroid plexus epithelial cells (CPECs) with high purity in vitro from neonate rat dissociated by mechanical method without enzyme digestion. We have demonstrated, for the first time, that neural stem cells exist in the rat choroid plexus and the number of neural stem cells is age-dependent. In our study, the transplanted CPECs were found to survive and spread in the perihematoma areas, improving the recovery of neural function that highly disables the suffers, and promoting the growth of regenerating axons in the model of rat intracerebral hemorrhage. Grafting of CPECs and neoplastic neuronal cells were observed in the hematoma areas. In addition, the volume of the hematoma was found to exhibit the most significant reduction. However, there is no report about CPECs transplantation in experimental SCI. This study will be conducted to corroborate the hypotheses that CPECs can promote nerve regeneration when grafted into SCI lesions. CPECs will be isolated mechanically and cultured from neonatal rat, encapsulated within alginate microcapsules, and grafted into SCI lesions. After surgery, animals will be periodically tested for evoked potentials and motor function by Basso-Beattie-Bresnahan scale. Molecular biology methods, such as immunohistochemistry analyses, western blotting and real time-PCR, will be performed. The expected findings may indicate that CPECs have the ability to facilitate axonal growth in vivo, suggesting that they may be a promising candidate as graft for the promotion of nerve regeneration in the spinal cord. Additionally, we will investigate mechanisms of nerve regeneration that molecules of the canonical Wnt signaling pathway may have direct effects on axonal growth in SCI through neurotrophin signaling. Our study is aimed at exploring the potential clinical application of cell transplantation in the treatment of SCI, and further providing new insights into axonal growth in SCI.

脊髓损伤（SCI）是一种高致残性损伤，高致残率给社会和家庭带来了巨大的负担，而目前对SCI后神经功能损害尚无确切有效的治疗手段。近年来，脉络丛移植对中枢神经损伤的修复作用引起了人们的关注。本课题组在前期的研究中首次建立了一种简便而实用的大鼠脉络丛上皮细胞（CPECs）的原代培养方法；首次发现大鼠脉络丛组织中的神经干细胞数量与鼠龄之间存在依赖关系；证实CPECs移植治疗脑出血可促进轴突的再生和新生神经元的产生，缩小血肿。但CPECs移植治疗SCI国内外尚无报道。据此，本课题组提出CPECs对脊髓损伤同样具有神经修复作用。为证实该假说，本研究拟在建立大鼠SCI模型的基础上，运用微囊技术包裹CPECs，采用行为学观察以及现代分子生物学等实验方法，从体内外两方面揭示CPECs对脊髓损伤的神经修复作用，并从神经营养因子角度揭示CPECs对SCI神经修复的信号转导通路，为细胞移植治疗SCI提供新思路。

脊髓损伤是一种严重的临床疾病，影响着患者的生活质量。在脊髓损伤后，自身修复能力有限，而移植的外源细胞通过局部分泌活动改善受损区的微环境，故细胞移植作为一种有前景的治疗脊髓损伤及病变的方法受到越来越多的重视。本项目重点观察了脉络丛上皮细胞（CPECs）对脊髓损伤的神经修复作用。主要研究内容包括：通过酶联免疫吸附法（Elisa法）检测不同条件下CPECs分泌的神经营养因子的变化，CPECs移植对脊髓损伤后神经修复的观察，神经营养因子与细胞信号转导途径的关系。主要研究结果包括：（1）建立了简单易行的种子细胞制备方法；（2）发现了原代培养的种子细胞中含有祖细胞并能分化；（3）明确了微囊化对脉络丛上皮细胞分泌功能的影响；（4）采用磁共振扩散张量成像(DTI)技术确立了大鼠脊髓横切模型更适用于脊髓损伤的神经再生研究；（5）采用DTI技术确定了脊髓损伤后最佳检查时机，即脊髓损伤后24小时为大鼠脊髓损伤的最佳检查时机；（6）确定了脊髓损伤的最佳电生理检测方法，即体感诱发电位和脊髓诱发电位是脊髓损伤的敏感检测方法；（7） 明确了不同培养条件和方式对种子细胞分泌功能的影响；（8）证实了CPECs对脊髓损伤的神经修复作用。本项目结果表明CPECs对脊髓损伤的神经修复作用，为CPECs移植治疗SCI提供了部分新的理论和实验基础。