宫内羊膜腔注射移植的干细胞向神经管畸形病变部位定向迁移的分子机制研究及其远期神经功能恢复的效果评价

Neural tube defects (NTDs) are the second most common congenital anomalies currently with no effective treatment. It is of great significance to explore effective treatments for NTDs during eary development of embryos. Our previous studies showed that intra-amniotic mesenchymal stem cells (MSCs) transplantation to NTDs at early development achieved beneficial functional recovery. MSCs injected into the amniotic cavity spontaneously migrated into the defective neural tissues. Engrafted MSCs specifically differentiated into cell types of defective tissues including skin stem cells, muscle cells, neural stem cells, neurogliocytes, and different types of neurons in situ, and futher triggered skin-lesion repair, neural functional recovery. To harness the potential of directional migration of MSCs, an understanding of the molecular mechanism is needed. MSCs express a broad range of chemokine and growth factor receptors, which suggests that the soluble ligands of these receptors may play a role in MSCs migration. Here, we aimed at testing our hypothesis that the interaction between the MSC surface receptors and molecules in the defective tissue may be responsible for the NTD lesion-specific migration. This study will screen the potential factors involving in the lesion-specific migration in the MSCs and defective neural tissue using multiple high throughput screening technologies. For improving the therapeutic effect of intra-amniotic MSC administration, we will cause these potential factors to over-express in MSCs and defective neural tissue by gene transfection and perform combined transplantation of MSCs and neural stem cells (NSCs), which should greatly facilitate the migration of MSCs toward the deformed neural tube and promote directional differentiation of MSCs into neurons. The best treatment scheme was selected by comprehensive analysis of the above treatment combination. Finally, the treatment will be performed in large animal models to evaluate the efficacy and safety of intra-amniotic stem cell transplantation. After stem cell treatment, the long-term follow-up observation and neurological function evaluation will be performed, which will provide the primary foundation for the following clinical trials.

神经管畸形（NTDs）是常见先天性畸形，目前尚无满意的产前治疗方法。在前期系列研究的基础上，我们首次发现胎鼠羊膜腔注射移植的骨髓间充质干细胞（MSCs）可以定向迁移到NTDs的脊髓缺损部位，并分化为不同种类的细胞参与组织修复。为深入研究其分子机制，本课题提出如下假说：即MSCs和病变脊髓组织分别高表达具有相互作用的因子，通过两者之间相互作用促使MSCs发生定向迁移。因此，本课题将采用多种组学联合检测和生物信息学贯通分析的技术筛选并验证参与定向迁移的相关因子。然后通过转基因方法使迁移相关因子高表达以促进MSCs定向迁移，并通过与神经干细胞联合移植促进MSCs向神经细胞分化，最后综合分析不同治疗组合的效果确定羊膜腔注射移植干细胞的最佳治疗方案，在兔和羊的NTDs模型进一步验证治疗效果，并进行生后长期随访和下肢、膀胱、肛门直肠的神经功能恢复评定，为将来进行临床试验提供有价值的前期研究基础。

神经管畸形（Neural tube defects, NTDs）是环境因素和遗传因素共同作用的复杂多基因遗传疾病，发病机制尚不清楚，目前开展的胎儿外科治疗仍无法恢复神经功能障碍。本课题组在前期研究发现NTDs胚胎期就有明显运动和感觉神经元数量减少的基础上，提出神经元修复重建的治疗新思路。本课题发现神经分化相关的神经营养因子表达量随着胎龄增加逐渐降低，而且NTDs胎鼠脊髓内的神经营养因子表达明显减低，其中BDNF表达降低非常明显。我们将腺病毒包装的BDNF以及联合BMSC进行羊膜腔注射治疗，治疗后皮肤缺损面积明显缩小，BMSC定植区域细胞凋亡明显减少，凋亡相关基因明显改善，移植BMSC可分化为成熟的感觉、运动神经元。利用全胚胎体外培养发现BMSC可自主迁移到NTDs的缺损部位。进一步研究发现HGF/c-Met相互作用是促进BMSC自主迁移的分子机制。建立转基因CRMP4 siRNA与BMSC联合移植治疗方法，既可以对NTDs病因进行靶向治疗，也可以利用BMSC对缺损组织修复重建，联合治疗的效果比单一治疗效果明显。利用蛋白质组学检测NSC源外泌体中蛋白质，发现Netrin1富集表达于NSC外泌体中，Netrin1通过上调Hand2、Phox2b促进BMSC向神经元分化。利用蛋白质芯片对羊膜腔内移植BMSC的孕鼠羊水进行检测，发现BMSC移植明显改善羊水微环境，其中ACTIVIN A、NGF、BDNF、CNTF和CXCR4是微环境改善的关键因子，是提高干细胞移植治疗NTDs效果的最佳基因干预靶点。利用NTDs动物模型发现miR-322通过调控NOX4参与NTDs发生，羊膜腔内注射miR-322 mimic治疗后细胞凋亡减少，是有应用前景的治疗靶点。另外，利用NTDs胎儿标本、动物模型和各种组学技术筛选和验证，发现Bhlhe40、Sirt1、LMNA、CORO1A、DNM2、C3和C9是NTDs致病基因。本课题的研究已发表标注课题资助的论文24篇，其中SCI收录论文22篇（其中中科院分区1区5篇，2区12篇），中文论文2篇。申请发明专利6项，已获批4项，其中1项于2020年获得成果转化，转化经费100万元。相关研究成果获得辽宁省科技进步一等奖1项，中国医院协会医院科技创新三等奖1项。本课题培养7名博士研究生和2名硕士研究生。