整合素β1与细胞外基质蛋白相互作用调控间充质干细胞向神经管畸形组织定向迁移的作用机制研究

Neural tube defects (NTDs) are severe congenital anomalies of the central nervous system that result from incomplete neurulation. However, foetal surgical repair of the defects did not improve neurological outcome of patients. In previous study, we successfully devised a prenatal treatment and transplanted mesenchymal stem cells (MSCs) into amniotic cavity of embryos at early stage of NTDs formation, and we found transplanted MSCs specifically surived in defective region of neural tube. And surived MSCs could differentiate into neural stem cells, neurogliocyte and neurons, which reveals the possibility of intra-amniotic MSCs injection for prenatal management of NTDs. However, compared to large defectively developed neural tube tissue, there was relatively less regenerated neurons and tissue repair, thus we intended to find out new strategy to enhance the ability of MSCs to migrate toward nerve injury site. We performed an expression screening using RNA-seq technique and discovered the expression of several chemotatic factors and molecules and neurotrophins is altered in the deformed cranial neural tube, and the changes in expression of extracellular matrix molecule –LAMB3, LAMC2 and COL4α were noticeably. So we speculate that these genes may play a critical role for the migration of MSCs toward the defected neural tube. LAMB3, LAMC2 and COL4α participates in numerous cellular processes including cell attachment and spreading, migration and proliferation via interacting with integrinβ1 ( ITGB1). Based on these, in this project, we intend to treat NTDs with intra-amniotic administration of MSCs transfected with ITGB1 on early development stage. MSCs transferred with ITGB1 might greatly facilitate the migration of MSCs toward the deformed neural tube, and could protect survival and differentiation of transplanted MSCs in the neural tissue. Intra-amniotic ITGB1-MSCs administration might greatly promote the functional recovery of neural tube defects.

神经管畸形(NTDs)是常见的严重先天畸形，尚无良好治疗方法。在前期研究基础上，我们建立了胚胎早期羊膜腔内干细胞移植技术，发现间充质干细胞（MSCs）具向神经管畸形部位定向迁移的能力且能够修复脊髓缺损组织。利用RNA-seq，我们发现多个细胞外基质蛋白—LAMB3、LAMC2和COL4α在脊髓缺损部位的高表达。本课题提出新假说：早期NTDs胚胎畸形组织中ECM蛋白过表达，通过与MSCs表面受体整合素β1相互作用，引导MSCs定向迁移至畸形部位，促进NTD的修复。为验证上述假说，本课题将利用体外培养的胚胎和细胞，研究ITGB1/ECM蛋白对MSCs的定向迁移的相互作用及分子机制。利用NTDs动物模型研究通过羊膜腔注射方法移植过表达/敲除ITGB1的MSCs，对过表达/敲除ECM蛋白的NTDs胚胎进行治疗，观察其治疗效果，探讨在严重结构异常或神经不可逆损伤发生前对NTDs修复治疗的新方法。

神经管畸形(NTDs)是常见的严重先天畸形，目前尚无良好治疗方法。在前期研究中，我们发现神经元发育在NTDs发生早期便存在异常。因此，我们提出在胚胎早期挽救神经元死亡或进行神经元修复重建是治疗NTDs神经功能损伤的关键。在本研究中，我们利用体外NTDs胚胎模型建立了羊膜腔内干细胞移植技术，发现间充质干细胞（MSCs）具向神经管畸形部位定向迁移的能力，利用RNA-Seq测序筛查到ITGB1/ECM分子、HGF/c-Met参与了MSCs向NTDs缺损区域的定向迁移。利用体内NTDs胎鼠模型，我们发现羊膜腔内移植的MSCs可以转分化为皮肤细胞和不同类型的神经元以替代损伤的组织细胞，修复缺损的皮肤组织并促进神经功能修复。进一步，我们在发现CRMP4 异常高表达引起细胞凋亡是神经管畸形发生的关键原因的基础上，建立了羊膜腔内MSCs移植与基因转染CRMP4 siRNA联合治疗NTDs的新方法。既可以对NTDs病因进行靶向治疗，也可以利用MSCs对缺损组织修复重建。结果显示CRMP4可作为治疗NTDs的一个新的治疗靶点，降低CRMP4在脊髓中的表达，可以改善移植的MSCs周围微环境，提高MSC移植治疗脊柱裂的效果。另外，我们发现羊膜腔内移植的MSCs除了在畸形部位定植，也在羊水与羊膜中存活，我们利用蛋白质组学对羊水中的细胞因子进行检测，结果发现32个细胞因子明显在MSCs移植后明显高表达。根据生物信息学分析和差异蛋白表达量变化情况，我们选择神经营养因子ACTIVIN A、NGF、BDNF、CNTF和细胞迁移因子CXCR4进行深入研究。干预实验表明，上调神经营养因子ACTIVIN A、NGF、BDNF、CNTF可抑制胚胎脊髓神经凋亡，促进突触发育。抑制这些因子的活性减弱移植MSCs的抗凋亡和促分化作用。抑制CXCR4活性可降低MSCs在NTDs胚胎神经管中的定植率。这些因子是提高MSCs移植治疗NTDs效果的最佳基因干预靶点。以ITGB1/LAMC2为分子靶点联合MSCs移植增强了MSCs的迁移能力，提高ITGB1/LAMC2的表达后，明显促进了MSCs在NTDs体外胚胎畸形部位的定植率，提高了MSCs对NTDs胎鼠的皮肤修复能力。本课题的研究已发表标注课题资助的SCI收录论文7篇（其中中科院分区1区2篇，2区4篇）。