干细胞移植在胚胎组织内的迁移、分化规律及其对隐性脊椎裂的治疗效果研究

项目名称： 干细胞移植在胚胎组织内的迁移、分化规律及其对隐性脊椎裂的治疗效果研究

项目编号： No.30872705

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 袁正伟

作者单位： 中国医科大学

项目金额： 35万元

中文摘要： 随着产前诊断技术的不断发展，越来越多的神经管畸形可以在产前早期诊断，但是目前尚无良好干预治疗方法。在前期研究发现先天性脊椎裂胎鼠的脊髓神经元存在明显发育异常的基础上，提出干细胞移植创建的新治疗思路。本课题成功建立胎仔外科、显微外科和显微注射相结合的干细胞移植技术，将GFP标记的MSCs分别移植到16天、17天和18天脊椎裂胎鼠的病变脊髓处，发现绿色荧光标记的干细胞可以在病变的脊髓内存活，并可分化为神经干细胞标志物、胶质细胞、神经元等不同神经细胞,EGF和FGF8在干细胞迁移中起重要的作用。同时发现移植MSCs在局部脊髓组织内不仅自身表达神经营养因子，而且可以诱导周围组织细胞表达这些神经营养因子，凋亡细胞明显减少。利用逆向神经示踪方法发现移植的MSCs与椎旁肌肉建立了突触联系。关于先天性脊椎裂胚胎发生机制研究方面，发现先天性脊椎裂胎鼠腰骶段脊髓的神经上皮组织中细胞凋亡明显增加，细胞增殖明显减少。利用双向电泳和质谱方法发现13种新的与先天性脊椎裂发生相关的差异表达蛋白质，这些蛋白质参与细胞凋亡发生和内质网应激反应，这些新发现对神经管畸形发生机制的探索提供了新的线索。

中文关键词： 先天性脊椎裂；间充质干细胞；宫内细胞移植；神经元

英文摘要： Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this project, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to fetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord,intrinsic EGF and FGF8 might play important roles in MSCs migration. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTDs. With retrograde tracing using fluro-gold, we found some transplanted MSCs were labelled with fluro-gold indicating that transplanted MSCs established synaptic connections with muscles, this suggested that transplanted MSCs differentiated into functionally active neurons.In the studies of embryogenesis of NTDs, we observed increased cell apoptosis and decreased proliferation in developing neuroepithelium of lumbo-sacral neural tubes. Using 2-D gel electrophoresis and mass spectrometry, we found 13 proteins were defferentially expressed in rat fetuses with spina bifida aperta, these proteins participated in cell apoptosis and endoplasmic reticulum stress, our new finding provided new hints for understanding the pathogenesis of NTDs.

英文关键词： neural tube defects; mesenchymal stem cells; in utero cell transplantation;neuron