TG2/SHH基因修饰EMSCs-Fibrin支架对NSCs命运调控机制及修复脊髓损伤研究

As a well-known morphogen, SHH plays an important role in regulating the development of nervous system. It can promote NSCs’ proliferation and induce their differentiation as well. However, the molecular mechanisms of SHH’s dual effects are yet to be clarified. Based on previous reports and our preliminary experimental data, we hypothesize that the free SHH may promote the proliferation of NSCs via the classical signaling pathway (Patched-Smoothened-Gli), while SHH multimers, cross-linked to extracellular matrix, can activate the non-canonical BOC-SFKs pathway and Integrins-FAK pathway to induce the differentiation of NSCs. This project focuses on using TG2 and SHH genes to modify the nasal mucosa-derived EMSCs which will be cultured in the fibrin glue. These EMSCs will secrete the cross-linker TG2 and bioactive factor SHH in the fibrin. Thus forming a novel, bioactive SHH-Fibrin scaffold featuring autocrine and self-assembly. The outcome of the investigations will reveal the effects exerted by this SHH-Fibrin scaffold in regulating the embryonic rat spinal cord NSCs as well as the underlying molecular mechanisms. Afterwards, the transgenic EMSCs/Fibrinogen hybrid gel will be injected into the spinal cord lesions to form SHH-Fibrin self-assembly scaffolds in situ, followed by the evaluation of the scaffolds’ impact on promoting proliferation, migration and differentiation of the endogenous NSCs, and their effects on the establishment of neural relay network to repair the spinal cord injury. This project provides a new strategy for the application of stem cells/scaffold which could offer a great potential in improving the repairing of the injured spinal cord.

SHH是调控神经系统发育的关键形态发生因子，既能促进NSCs增殖又能诱导NSCs分化。然而，其双重调节的分子机制仍不太清楚。根据文献报道和前期的预实验结果，我们推测：游离SHH可能通过经典Patched-Smoothened-Gli通路促进NSCs增殖，而交联于细胞外基质的SHH多聚体将激活非经典BOC-SFKs通路并协同Integrins-FAK通路诱导NSCs分化。本项目将TG2和SHH基因修饰的EMSCs共同种植于Fibrin胶，通过EMSCs持续分泌交联剂TG2和生物活性因子SHH，构建自分泌、自交联的SHH-Fibrin生物活性支架；观察支架对大鼠脊髓NSCs命运的影响，并探讨其分子机制；将转基因EMSCs/Fibrinogen混合溶胶注入脊髓损伤部位，原位自组装成SHH-Fibrin支架，评价其促进内源NSCs有序增殖、迁移和分化，建立神经接力网络修复脊髓损伤的效果。

干细胞和细胞外基质移植修复脊髓损伤具有重要的科学研究价值和临床应用前景。本项目旨在将TG2和SHH基因修饰的鼻黏膜来源的外胚间充质干细胞（ectodermal mesenchymal stem cells，EMSCs) 共同种植于Fibrin胶，构建一种生物活性支架，将支架移植入脊髓损伤部位，评价其修复脊髓损伤的效果。本课题完成了以下研究内容并取得了预期的研究结果：（1）EMSCs生物学特性表明EMSCs是一种多能干细胞，可诱导分化为神经细胞、神经胶质细胞和成骨细胞，是一种比较理想的用于移植修复神经系统损伤的种子细胞。（2）重点研究了EMSCs对NSCs向神经细胞和神经胶质细胞分化的分子调节机制。研究结果表明，EMSCs可作为一种活的生物材料为NSCs的分化和生长提供良好的全能的（“All in One”）微环境。该微环境由ECM; 生长因子；细胞连接和物理模量组成。这种微环境可促进神经干细胞向神经细胞分化而抑制其向星形胶质细胞分化。与此同时，该微环境可诱导神经干细胞的脂筏组装，进而募集各种有利于神经元分化的信号接受分子（受体）进入细胞膜的脂筏内，以构筑一个与微环境相应的“All in One”信号感受平台，协调一系列信号通路的信号转导过程，促进神经干细胞向神经元定向分化，同时驯化星形胶质细胞的生物学行为。（3）研究了SHH以及TG基因基因修饰对EMSCs分化的影响。结果表明TG 或SHH过表达可促进EMSCs向神经细胞和少突胶质细胞分化。（4）构建一种含有SHH基因重组腺病毒的Fibrin生物活性支架。将NSCs 种植于该支架，可向神经细胞分化。（5）用TG-EMSC和SHH-EMSC作为双种子细胞与Fibrin胶构建了SHH 缓释放支架，该支架具有明显的促进神经干细胞分化和神经纤维生长的作用；将EMSCs/支架移植入脊髓损伤部位，可促进脊髓损伤修复。本项目的研究结果为干细胞和细胞外基质移植修复脊髓损伤提供了一种理想的种子细胞和构建组织工程支架的新理念。该理念的核心是生物活性支架可提供种子干细胞、ECM和神经细胞分化所需要的生长因子，使支架既能促进神经干细胞分化又能驯化星形胶质细胞的生物学行为，以促进神经有序再生。本研究为干细胞的临床应用提供了实验研究基础。