Bcl-2过表达及其亚细胞定位对BMSCs移植治疗大鼠脊髓损伤的影响及其机制研究

Bone marrow stromal stem cells (BMSCs) transplantation for spinal cord injury (SCI) has been shown to improve functional recovery. However, whether BMSCs can directly promote axonal regeneration and myelination is still unclear, and the therapy is limited by the low survival rate and low neural differentiation rate of BMSCs after transplantation. Bcl-2 can promote cell survival and neural differentiation, and support axonal regeneration through its specific subcellular localization. Our previous studies have found that up-regulation of Bcl-2 is closely related to the efficacy of BMSCs. We hypothesize that overexpression of Bcl-2 can promote the survival and neural differentiation of BMSCs after transplantation for SCI, and activate Ca2+-ERK/CREB pathway through its specific subcellular localization to support axonal regeneration. In this project, BMSCs overexpressing Bcl-2 with different subcellular localization will be constructed and transplanted for SCI. Immunofluorescence staining, Western blot, neural tracing and behavior tests will be used to detect the survival and neural differentiation of BMSCs, axonal regeneration, myelination, functional recovery and the activation of the Ca2+-ERK/CREB pathway in the injured spinal cord after BMSCs transplantation, in order to verify the effects of Bcl-2 overexpression and subcellular localization on BMSCs transplantation for SCI and their underlying mechanisms, and to provide new theoretical basis and technical support for clinical treatment of SCI.

骨髓基质干细胞(BMSCs)移植治疗脊髓损伤(SCI)能促进神经功能恢复。但BMSCs能否直接促进轴突再生和髓鞘形成尚未明确，且还存在着移植后存活率和向神经细胞分化率较低的问题。Bcl-2能促进细胞存活和神经分化，并可通过亚细胞定位促进轴突再生。前期研究发现BMSCs的功能发挥与Bcl-2上调密切相关。课题组假设：过表达Bcl-2能促进BMSCs在SCI移植体内存活和向神经细胞分化，并能通过亚细胞定位激活Ca2+-ERK/CREB通路促进轴突再生。本课题拟构建过表达不同亚细胞定位的Bcl-2的BMSCs，移植治疗SCI。利用免疫荧光染色、免疫印迹、神经示踪和行为学等技术检测移植体内BMSCs的存活、分化、轴突再生、髓鞘形成和功能恢复，以及Ca2+-ERK/CREB通路的活化情况；以验证Bcl-2过表达和亚细胞定位对BMSCs移植治疗SCI的影响及其机制，为临床治疗SCI提供新的依据和方向。

脊髓损伤导致髓磷脂碎片产生，炎症持续，胶质瘢痕和纤维瘢痕形成，阻碍了神经再生，也抑制了骨髓基质干细胞(BMSCs)的移植治疗效果。本项目针对：1) BMSCs移植治疗脊髓损伤后存活率低和以及死亡原因尚不清楚，2)脊髓损伤后胶质瘢痕和纤维瘢痕形成机制尚未阐明，以上两个问题进行了深入研究。目前主要取得如下研究进展：(1) 过表达Bcl-2的BMSCs移植能对抗髓磷脂碎片和M1炎症诱导的细胞凋亡，促进脊髓损伤功能恢复；与预期不同的是，过表达定位于内质网的BCl-2的BMSCs反而丧失了抗凋亡特性，并不能对抗髓磷脂碎片和M1炎症诱导的细胞凋亡 [Bcl-2部分结果发表在Neural Regen Res, 2019, 14(10): 1765-1771]。(2) 在脊髓损伤病理机制方面，我们发现了肌成束蛋白Fascin-1在脊髓损伤后的小胶质细胞中特异性高表达，并调控小胶质细胞迁移，其可能在小胶质细胞瘢痕的形成中发挥关键作用 [该成果发表在Front Pharmacol, 2021, 12: 729524]。(3) 证明了脊髓损伤中，M1极化的小胶质细胞通过TGFβ1/SOX9通路促进星形胶质细胞产生CSPG，从而影响星形胶质瘢痕的形成 [该成果发表在Neural Regen Res, 2022, 17(5): 1072-1079]。(4) 初步证实了脊髓损伤后M2型巨噬细胞能通过PDGFB/PDGFRβ通路促进成纤维细胞迁移，从而形成纤维瘢痕 [该成果发表在Front Pharmacol, 2021, 12: 670813]。在此基础上，我们将进一步开展BMSCs移植对如上病理过程的影响及其机制的研究，为脊髓损伤的特异性分子靶向和干细胞移植的联合治疗提供基础和依据。