移植的大鼠脉络丛上皮细胞与器官型脊髓组织片相互整合及相关机制

Spinal cord injury（SCI） is a disease with a high rate of disability, and its high disability rate often results in high social and economic burden. At the present time, there is no effective medical therapy available to restore function in patients with SCI. Recently, the transplantation of the choroid plexus epithelial cells (CPECs) has been tested to impart neuroprotection against central nervous system disease in several animal models. The role of CPECs in growth factor and nutrient production makes it a novel potential source of transplantation. We have established the high purity CPECs in vitro from neonate rat dissociated by mechanical method without enzyme digestion, which is a simple and applied way of primary cell culture. We firstly demonstrate neural stem cells exist among rat choroid plexus by immunohistochemistry, and the change of neural stem cells is age-dependent. Transplanted CPECs can survive and distribute in SCI lesions, improve functional recovery, and promote the growth of regenerating axons in the model of rat SCI. We firstly demonstrate the primary cultures from choroid plexuses contain progenitor cells that differentiate into choroid plexus epithelial cells in vitro. The protective role of CPECs in SCI is proved, but the underlying mechanism of the protection is not fully understood. Further studies based on our findings are needed to better understand the effect of CPECs and the relevant mechanism. This study will be conducted to corroborate the hypotheses that the transplanted CPECs and SCI interact with each other to form a new system, which promotes nerve regeneration. We will carry out the present study that CPECs will be isolated mechanically and cultured from neonatal rat, and grafted into cultured organotypic spinal cord slice. The molecular biology methods, such as enzyme-linked immunosorbent assay, immunohistochemistry analyses, and western blotting will be further performed, because they provide a unique tool for the study. Basing on these, we will associate organotypic spinal cord slices with grafted CPECs to observe the inducing effects of spatial signals from organotypic spinal cord slices on transplanted cells biological behaviors. Then, we will analyze the alteration of organotypic spinal cord slices modified by CPECs on the aspects of host cells behaviors. Further, we will observe the new system formed by transplanted CPECs and host tissue through their cell-cell connection, cell types and intracellular signal. In order to research the effects of affected microenvironment on grafted CPECs, the oxygen - glucose deprivation model in organotypic spinal cord slice will be established. We may observe the cell behavior alterations of transplanted CPECs and host tissue using same methods as mentioned previously to analyze related integration mechanisms. The results of our study suggest the potential clinical applications of cell transplantation treatment in SCI, providing new insights into SCI.

脊髓损伤(SCI)是一种高致残性损伤，目前对SCI尚无有效的治疗方法。近年来，脉络丛上皮细胞（CPECs）移植治疗中枢神经损伤引起了人们的关注。本项目组在前期的研究中建立了一种简便易行的大鼠CPECs的培养方法；发现大鼠脉络丛中的神经干细胞数量与鼠龄之间存在依赖关系；原代培养的CPECs中含有Nestin阳性细胞；以及证实CPECs移植治疗SCI可促进轴突的再生和新生神经元的产生。尽管研究证实CPECs移植对SCI具有治疗作用，但其作用机制还不明了。据此，本项目组提出移植的CPECs和宿主组织通过相互整合而发挥神经修复作用。为证实该假说，本研究拟采用器官型脊髓组织片培养方法，构建氧糖剥夺损伤模型，将CPECs移植至脊髓组织片，采用现代分子生物学实验方法，分析移植细胞、宿主以及两者所形成的"整合体"生物学行为变化，揭示CPECs对SCI神经修复的可能作用机制，为细胞移植治疗SCI提供新思路。

脊髓损伤严重影响着患者的生活质量，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）与器官型脊髓损伤组织片的整合作用。主要研究内容包括：（1）建立器官型脊髓组织片的培养液配置、正常器官型脊髓组织片培养模型的培养方法以及器官型脊髓OGD模型的培养方法，（2）评估移植的CPECs与器官型脊髓组织片的相互整合变化。主要研究结果包括：（1）建立了配方简单的大鼠器官型脊髓片培养液的配制方法，（2）建立了简单易行的正常培养器官型脊髓组织片模型，（3）构建了大鼠器官型脊髓组织片的OGD模型，（4）明确了不同培养方式对CPECs分泌功能的影响，（5）观察了CPECs移植于正常培养的大鼠器官型脊髓组织片模型的变化，（6）评估了CPECs移植于大鼠器官型脊髓组织片OGD模型的变化，（7）确定了标记的CPECs移植于大鼠器官型脊髓组织片OGD模型的整合变化，（8）提出了急性脊髓损伤DTI检查的最佳时机和生物标记物。本项目结果表明CPECs与器官型脊髓组织片可形成整合体，发挥神经修复作用，为CPECs移植治疗SCI提供了部分新的理论和实验基础。