利用再生模式生物蝾螈(Ambystoma mexicanum)研究启动脊髓再生的机制

While spinal cord injury is permanent and irreversible in mammals, axolotl can fully regenerate the injured spinal cord on both cellular and functional level. This uniquely positions the axolotl as the ideal model for dissecting the mechanism behind spinal cord regeneration. Removal of a piece of tail spinal cord inhibits axolotl tail (including spinal cord) regeneration, indicating spinal cord harbors key cell types and factors initiating regeneration. Our unpublished preliminary results identified that the depletion of neurons results in a failure to induce spinal cord and tail regeneration. These results suggest that there is a dependency on neurons and neuronal factors in initiating tail and spinal cord regeneration. ..To further understand the cellular and molecular dependency on neurons in regeneration, we will start by establishing transgenic axolotls. Taking advantage of characterized tissue-specific promoters and recently established knock-in approaches, we will label and manipulate NSCs and neurons, separately. Furthermore, we will, (1) observe the interaction between NSCs and neurons during spinal cord regeneration using fluorescence live-imaging; (2) study spinal cord regeneration by removing the pools of NSCs or differentiated neurons, in a defined region of transplanted spinal cord using Nitroreductase. This allows us to dissect whether NSC proliferation and migration show a neuronal dependency during regeneration. Our study will thus provide a theoretical and practical basis for improving the limited capacity of mammalian spinal cord regeneration.

成年哺乳动物不能修复受损的脊髓。而蝾螈可在细胞及功能水平上完全再生损伤脊髓，为研究脊髓再生机制的理想模式生物。移除尾部脊髓可抑制蝾螈的断尾（包括脊髓）再生，表明脊髓包含启动再生的重要细胞类型及因子。我们尚未发表的初步研究表明神经元可能为启动再生的关键。我们将利用神经干细胞及神经元的特异表达启动子建立一系列转基因蝾螈，进一步 (1) 通过荧光实时成像观察脊髓再生中神经干细胞与神经元的互作；(2) 结合脊髓移植技术，通过激活自杀基因Nitroreductase，进而研究其再生。试图揭示脊髓再生中神经干细胞的增殖与迁移是否依赖于神经元轴突的生长等关键问题。研究将为改善哺乳动物损伤脊髓修复提供依据。

脊髓损伤修复是当前医学及生物学领域面临的重大难题。本项目以中枢神经系统可再生的模式生物墨西哥钝口螈为模型，研究：（1）建立及优化墨西哥钝口螈活体及脊髓成像技术；（2）在墨西哥钝口螈中建立及优化特异细胞类型诱导自杀技术；（3）并进一步利用所建立优化的方法初步探索脊髓再生的细胞生物学机制。我们利用所建立的转基因蝾螈模型，建立并优化了蝾螈脊髓活体成像条件。初步研究发现：在脊髓再生早期，神经元轴突与神经干细胞密切互作。沿脊髓生长方向，神经元轴突末梢先于神经干细胞，极有可能为脊髓再生神经干细胞的迁移探索并提供了物理轨道，并决定了再生脊髓的生长方向。此外，我们在蝾螈中建立并优化了基于 NTR-Mtz系统的特异细胞诱导自杀技术，并进一步利用此技术研究了脊髓神经干细胞在再生中的功能。初步结果表明在移除脊髓神经干细胞条件下，移植脊髓及周边组织再生阻滞。本研究为进一步深入利用蝾螈研究脊髓再生的分子及细胞生物学机制提供了重要手段。