抗凋亡基因修饰神经干细胞促进脑梗死修复的MR报告基因成像研究

Neural stem cells (NSCs)-based therapy is the most promising therapeutic strategy for the treatment of cerebral infarction. However, the low survival rate in the implanted sites caused by a large number of apoptotic cells after transplantation seriously decreases their therapeutic effect. The neural repair efficacy using stem cell transplantation in cerebral infarction should be improved urgently. On the other hand, in vivo MRI tracking of stem cells after transplantation based on direct labeling method was limited because the long term survival and differentiation of implanted stem cells could not be accurately monitored as expected. A long-term, accurate method used for MRI tracking the whole biological process after stem cell transplantation is highly desired. In this study, we will construct a lentivirus vector with FerritinH-Bcl2 gene overexpression where nestin enhancer/promoter element is set as the start switch. Rat NSCs will be genetically modified by using this FerritinH-Bcl2 gene overexpressed vector to confer the capacities of anti-apoptosis and MR visualization to NSCs. After the genetically modified NSCs transplanted to the infarct lesions in the animal model of focal cerebral ischemia injury, the feasibility, efficacy and biological safety of this genetic modification will be determined at the levels of in vitro cells and in vivo living animals. The therapeutic efficacy of genetically engineered NSCs for cerebral infarction will be assessed. The survival, location, migration and differentiation of transplanted NSCs will be dynamic observed by using in vivo MRI and optical imaging. In light of the multidisciplinary integration of molecular MR imaging and molecular biology, the aim of this project is to improve the neural repair efficacy of stem cells-based therapy and to establish a new means to in vivo stability and dynamic monitor the long-term biological processes of transplanted stem cells, and thereby to provide more experimental evidence to promote the clinical translation of the stem cells-based therapy.

神经干细胞（NSCs）治疗是脑梗死最有前景的新治疗策略，但NSCs移植后低存活率严重影响其治疗效果，干细胞治疗脑梗死的效果亟待提高；采用直接标记MRI活体示踪难以长期对干细胞的存活及分化进行监测，干细胞治疗急需一种能长期准确示踪干细胞移植后生物学过程的方法。本项目构建FerritinH-Bcl2过表达慢病毒载体，设置Nestin启动子/增强子作为启动元件，对大鼠NSCs进行抗凋亡及MR报告基因修饰后移植治疗大鼠局灶性脑梗死，从细胞和动物整体水平考察双基因修饰NSCs的可行性、效率及安全性，观察基因工程化NSCs对脑梗死的修复作用，利用MRI及生物荧光成像活体动态观察NSCs移植后在体内的存活、定居、迁移及分化情况。通过MRI分子影像学、分子生物学等多学科交叉融合，探索提高干细胞治疗疗效及对干细胞移植后生物学过程进行长期稳定活体动态监测的新方法，为干细胞治疗临床转化提供更多实验依据。

神经干细胞（NSCs）移植虽能用于治疗脑梗死，然而梗死脑组织的微环境不利于干细胞存活，可导致干细胞移植后短期内大量凋亡，从而影响其治疗效果，也限制了干细胞的临床转化。B细胞淋巴瘤/白血病-2（Bcl2）基因是广泛存在于多种细胞中一种原癌基因，其编码产物Bcl2蛋白是细胞凋亡信号转导通路中的关键调节因子，属于能抑制多种原因引起的细胞凋亡的“抗凋亡基因”。 铁蛋白（Ferritin）普遍存在所有细胞中，铁蛋白重链（FerritinH）基因过表达可导致细胞内铁蛋白含量增高，在细胞内富集铁形成铁纳米粒子，使得细胞MRI可视化。FerritinH已作为一种MRI报告基因用于细胞长期活体MRI示踪。本项目利用基因工程技术，合成与构建了Nestin控制下的FerritinH-Bcl2条件过表达慢病毒过载体（Nestin-FerritinH-Bcl2）用于NSCs的MRI可视化及抗凋亡基因修饰，使得NSCs既能为MRI示踪又能上调Bcl2的表达。体外细胞试验显示，Nestin-FerritinH-Bcl2能对NSCs进行高效标记，标记率达70%以上，同时明显上调NSCs的Bcl2表达，提高了NSCs在乏氧环境下的生存率，而标记并不影响NSCs的干性及分化能力。活体动物实验表明，在大鼠局灶性脑梗死模型中，将Nestin-FerritinH-Bcl2标记的NSCs移植至脑梗死对侧纹状体后，标记的NSCs在体内的分布、迁移能被MRI活体动态监测直至移植后8周；Nestin-FerritinH-Bcl2标记的NSCs在移植后1周，其存活率较未标记对照组明显增高，移植后8周Nestin-FerritinH-Bcl2标记的NSCs分化成的神经元比例较未标记对照组高；移植Nestin-FerritinH-Bcl2标记的NSCs的大鼠其脑梗死面积及神经功能的恢复均优于对照组。本项目发展了一种Nestin控制下FerritinH和Bcl2基因修饰干细胞方法，能在对NSCs进行高效MRI标记的同时，对干细胞Bcl2抗凋亡基因进行有效上调，实现了在干细胞MRI长期示踪的同时对其抗凋亡能力的调控，提高了移植后干细胞生存率、神经元方向分化比例及其对脑梗死的治疗效果。本项目为提高干细胞治疗效果提供了示踪及凋亡性能联合调控的新策略，将有助于干细胞治疗在脑梗死中的临床转化，具有极大的应用潜力。