RNAi沉默NgR基因促进神经干细胞修复脑梗死的MRI分子影像学研究

Neural stem cell-based therapy is a promising therapeutic strategy in regenerative medicine, however, the efficacy of neural regenerative repair of cerebral infarction with stem cell transplantation need to be further improved. Based on the successful synthesis of a novel superparamagnetic iron oxide (SPIO)-loaded nanovesicle, we will develop a new class of cationic polymeric nanovesicles with superparamagnetic iron oxide naoparticele loaded in the inner cavity and NgR-targeted small interferon RNA (siRNA) bound to vesicle membrane as the drug nanocarrier to deliver siRNA and SPIO simultaneously into neural stem cells. Upon this multifunctional nanocarrier, neural stem cells will be epigenetically modified with use of RNA interferon as well as paramagnetically labeled with SPIO. After the treated cells are transplanted to the infarct lesions in the animal model of focal cerebral ischemia injury, cellular MRI will be used to in vivo, dynamic tracking the grafted, labeled cells. The efficacy of gene delivery, effect of cell labeling and biological safety of this siRNA-SPIO nanovesicle will be determine at the levels of in vitro cells and in vivo living animals. Its effect on the promoting neuronal differentiation of neural stem cells will be observed. The gene delivery to the cells and in vivo homing and migration of labeled cells will be detected by using MR imaging. The improved neural restorative effect of genetically stem cells on the repair of cerebral infarction will be determined. By virtue of molecular MR imaging, molecular biology and nanomedicine integration this project is aimed to establish a new means to improve the neural regenerative repair and neural function reconstruction as well as dynamic monitor the underlying process with neural stem cell transplantation, and thereby to advance the clinical translation of the stem cell-based therapy.

神经干细胞治疗是再生医学极有前景的新治疗策略，其促进脑梗死神经再生修复的效果亟待提高。本项目拟在已合成的高分子聚合物纳米囊泡基础上，制备携带靶向NgR基因的小干扰RNA及超顺磁性氧化铁纳米粒子的正电荷纳米囊泡作为输送载体，利用RNA干扰技术对神经干细胞进行表观遗传修饰，并进行细胞顺磁性标记，在局灶性脑梗死动物模型中进行移植干细胞的MRI活体动态监测，分别从细胞和动物整体水平考察携带小干扰RNA显像纳米囊泡的干细胞基因传输的效率、标记效果、生物安全性，观察基因修饰对促进神经干细胞向神经元方向分化的作用，并利用MRI监测基因传输及标记细胞在体内的分布及迁移情况，明确基因修饰用于干细胞提高脑梗死神经再生修复的效果。通过MRI分子影像学，分子生物学、纳米医药技术等多学科交叉融合，探索改善神经干细胞再生修复脑损伤、促进神经功能重建，并动态监测其过程的新方法，进一步促进干细胞治疗的临床转化。

脑梗死中移植的神经干细胞（NSCs）能在移植处存活，然而大多数分化成为胶质细胞，对神经元的替代作用有限。Nogo 受体复合体（NgR）被认为是影响神经干细胞向神经元分化的重要治疗靶点。对干细胞进行活体内的有效示综，仍是干细胞移植运用于临床面临的重要挑战。纳米高分子聚合物作为一种优良的载体，已广泛用于基因、药物及显像剂的传递。本项目合成了基于聚乙烯亚胺-聚乳酸的正电纳米囊泡，将水溶性SPIONs负载于囊泡水溶性空腔内，并通过静电作用负载带负电荷的NgR-siRNA，制备出同时负载SPIONs与NgR-siRNA的纳米复合物；在体外转染NSCs后，纳米复合物成功对干细胞进行磁性标记，同时高效地将siRNA转运至NSCs中，有效地沉默了NgR基因表达，提高NSCs向神经元分化百分比达到对照组的8倍。在大鼠脑梗死模型中，将转染后的GFP-NSCs移植至脑梗死同侧胼胝体中，同时负载NgR-siRNA及SPIONs的纳米复合物转染NSCs后，NSCs在体内的分布、迁移情况能被MRI动态、实时监测，组织免疫荧光染色检测表明转染NSCs向神经元分化百分比达到对照组的6倍，神经功能评分、MRI测量脑梗死面积表明对转染NSCs的神经功能修复效果显著优于对照组。本项目成功设计并合成了的同时负载SPIONs及siRNA的纳米复合物，实现了分子影像探针和核酸药物进行同步输送，在标记干细胞的同时，能对干细胞分化进行基因修饰和分化调控，不但实现MRI动态实时监测在细胞在体内定位、迁移情况，同时促进了移植干细胞向神经元方向的分化，提高了干细胞对脑梗死的治疗效果。这种集示踪与基因修饰于一体的干细胞治疗策略，在未来干细胞治疗脑梗死的临床实践中，具有极大的应用潜力。