缺氧靶向调控表达的NT-3基因修饰的神经干细胞移植治疗缺血性卒中的实验研究

Neural stem cells (NSCs) transplantation is a promising therapy for ischemic stroke. However, survival and neuronal differentiation of transplanted NSCs are so deficient that the therapeutic effect of NSCs transplantation for ischemic is unavailable. It has been reported that neurotrophin-3 (NT-3) gene modification can promote survival, proliferation and neural differentiation of NSCs. But the excessive expression of an exogenous gene may produce many deleterious side effects, such as tumorigenesis and epilepsy. In present project, we plan to construct a vector which can mediate hypoxia-targeted expression of NT-3. Under control of 5HRE, UTR and ODD, expression of NT-3 can be regulated at the transcription, post-transcription and post-translation levels specifically in response to hypoxia respectively. Then the neural stem cells modified with hypoxia-regulated neurotrophin-3 gene will be established. After transplantation of the gene modified NSCs into ischemic rat brain, hypoxia-targeted expression of NT-3 will be investigated and the effects of gene modification on survival, proliferation and neuronal differentiation of NSCs will be determined in vivo. Meanwhile, the effects of transplantation of the NSCs modified with hypoxia-targeted neurotrophin-3 gene on ischemic stroke in rats will be determined. The present studies will provide considerable rationale for NSCs-based transplantation therapy for ischemic stroke both in studies and clinical applications.

神经干细胞（NSCs）移植为缺血性卒中治疗提供了新的治疗策略，研究发现移植后NSCs的存活能力差、神经元分化比例少，达不到预期的治疗效果和目的。实验研究发现神经营养因子-3（NT-3）基因修饰可以促进NSCs的存活、增殖和神经元分化，但同时外源基因过度表达可能会引起肿瘤、癫痫等不良副作用。本项目通过构建缺氧靶向调控表达NT-3载体，用5HRE、UTR和ODD分别从转录、转录后和翻译后水平对NT-3的表达进行缺氧靶向调控，并获得缺氧靶向调控表达的NT-3基因修饰的NSCs。在大鼠局灶性脑缺血模型中，观察基因修饰的NSCs移植后NT-3的缺氧靶向性调控表达以及基因修饰对移植的NSCs存活、增殖及其向神经元分化的影响，同时观察基因修饰的NSCs移植对大鼠脑缺血的治疗作用。本课题的完成将为NSCs移植治疗缺血性卒中的研究及临床应用提供重要的理论依据。

神经干细胞（NSCs）移植为缺血性卒中治疗提供了新的治疗策略，研究发现移植后NSCs的存活能力差、神经元分化比例少，达不到预期的治疗效果和目的。实验研究发现神经营养因子-3（NT-3）基因修饰可以促进NSCs的存活、增殖和神经元分化，但同时外源基因过度表达可能会引起肿瘤、癫痫等不良副作用。本项目通过构建缺氧靶向调控表达NT-3载体，用5HRE、UTR和ODD分别从转录、转录后和翻译后水平对NT-3的表达进行缺氧靶向调控，并获得缺氧靶向调控表达的NT-3基因修饰的NSCs。在大鼠局灶性脑缺血模型中，观察基因修饰的NSCs移植后NT-3的缺氧靶向性调控表达以及基因修饰对移植的NSCs存活、增殖的影响，同时观察基因修饰的NSCs移植对大鼠脑缺血的治疗作用。本项目成功构建缺氧靶向调控表达的NT-3 载体质粒：pLV(Exp)-Puro-5HRE-SV40mp-NT3-ODD-UTR-sv40 pA-CMV-eGFP，并包装成缺氧靶向调控表达的NT-3 慢病毒：LV(Exp)-Puro- 5HRE-SV40mp-NT3-ODD-UTR-sv40 pA-CMV-eGFP，用其感染体外培养的大鼠胚胎皮质NSCs后获得缺氧靶向调控表达的NT-3基因修饰的NSCs：NSCs-5HRE-SV40-NT-3/ODD-IRES-EGFP-UTR（NSCs-5H-NT-3/ODD-UTR）。在NSCs-5H-NT-3/ODD-UTR组，缺氧后缺氧靶向调节系统可以上调NT-3基因mRNA和蛋白表达水平，且在体外培养的细胞缺氧损伤模型中发现5H-NT3/ODD-UTR基因转导减少缺氧诱导的NSCs凋亡。将NSCs-5H-NT-3/ODD-UTR移植入大鼠脑内，在线栓法制作的tMCAO模型3天后，与对照组相比，NSCs-5H-NT-3/ODD-UTR移植组脑组织中NT-3的表达显著上调（P<0.05），且NSCs-5H-NT-3/ODD-UTR移植治疗可以减小大鼠tMCAO后脑梗死体积和细胞凋亡（P<0.05），并减轻大鼠tMCAO后神经功能损伤。本课题的完成将为NSCs移植联合基因治疗缺血性卒中的研究及临床应用提供重要的理论依据。