胚胎期miR-9和miR-124体内转染联合干细胞移植治疗显性脊椎裂的研究

Spina bifida aperta (SBA) is complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with SBA have a multiple system handicap due to neuron deficiency of the defective spinal cord.In previous study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to fetal rat spinal column to treat retinoic acid induced SBA in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in SBA. Our results suggested that prenatal MSC transplantation might be an alternative strategy to treat spinal neuron deficiency in SBA by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord. However,compared to large defectively developed spinal tissue, there was relatively less regenerated neurons, thus we intended to find out new strategy to accelerate MSCs neural differentiation and host tissue neurogenesis. MicroRNAs are involved in numerous cellular processes including development,cell proliferation, differentiation. We investigated several miRNAs expression which are reported to be associated with neural tube defects, and observed continously decreased expression of miR-9 and miR-124 in fetal spinal cord with SBA from E12 to E15. miR-9 and miR-124 are also reported to play important roles in neurogenesis, and mediate fate conversion of human fibroblasts to neurons. Based on these,in this project, we intend to treat SBA with intraamniotic administration of miR-9 and miR-124 and in utero MSCs transplantation in early development stage. miR-9 and miR-124 administration could correct the disturbed expression of miRNA and their regulated genes, and also favorate the neural differentiation of transplanted MSCs. Combined technigue of miR-9 and miR-124 intraamniotic administration and in utero MSCs transplantation might greatly promote the functional recovery of SBA.

先天性脊椎裂是常见的先天畸形之一，目前尚无良好治疗方法。我们在前期研究中将骨髓间充质干细胞移植到脊椎裂胎鼠脊髓缺损部位，发现移植的干细胞能够在胎鼠脊髓中存活、迁移并分化成神经元，证明我们建立的胎仔外科和显微注射相结合的胚胎期干细胞移植技术是治疗脊椎裂非常有潜在应用前景的新方法。我们对脊椎裂胎鼠脊髓组织microRNA表达规律进行研究，发现miR-9和miR-124在脊椎裂胎鼠脊髓中的表达明显下调，证实其与脊椎裂的发生相关，同时它们的前体促进了干细胞向神经细胞的分化。因此，本课题在前期研究基础上，提出羊膜腔内转染miR-9 和/或 miR-124联合干细胞移植的方法对不同发育阶段的脊椎裂胎鼠进行综合治疗，通过恢复异常基因表达不仅可以消除脊椎裂发生的原因，而且还可以促进移植的干细胞对脊髓神经元修复重建，具有双重治疗作用，我们的研究对于探讨脊椎裂胚胎期治疗新方法具有重要意义。

先天性脊椎裂是常见的先天畸形之一，目前尚无良好治疗方法。我们在前期研究中将骨髓间充质干细胞移植到脊椎裂胎鼠脊髓缺损部位，发现移植的干细胞能够在胎鼠脊髓中存活、迁移并分化成神经元，证明我们建立的胎儿外科和显微注射相结合的干细胞移植技术是治疗脊椎裂非常有潜在应用前景的新方法。我们对脊椎裂胎鼠脊髓组织microRNA表达规律进行研究，发现miR-9和miR-124在脊椎裂胎鼠脊髓中的表达明显下调，证实其与脊椎裂的发生相关，并且两种miRNA 的表达下调可能与REST接到的BAF染色质重建复合物通路相关。同时它们的前体促进了干细胞向神经细胞的分化。本课题还进一步研究了胚胎期干细胞移植联合生长因子（EGF）转染治疗脊椎裂，证实联合治疗，增加了局部区域神经生长因子的表达，减少局部神经元的凋亡，比单独干细胞移植更有益于脊椎裂的治疗。课题组还研究了胚胎期和出生后移植干细胞对于干细胞神经分化潜力的影响，证实胚胎期移植更有益于间充质干细胞向神经方向分化。课题组重点研究了胚胎期miR-124移植对于脊椎裂的治疗作用，结果显示miR-124移植后，局部组织神经生长因子（NGF）的表达增加4-5倍，局部组织神经元凋亡明显减少。证实miR-124移植更有益于局部组织神经分化微环境的提升，对于脊椎裂治疗有重要的意义。