基于干细胞的新型靶向复合生物基因载体的构建与其对缺血性脑卒中的治疗研究

Mesenchymal stem cells (MSCs) have shown therapeutic potential in treatment of brain diseases, due to their capability of differentiation into neural cells under given conditions. Ischemic stroke which occupies 80% of cerebrovascular disease remains a leading cause of death and disability. Studies indicate the change of blood-brain barrier (BBB) caused by ischemic stroke provides premise of distribution of MSCs in the brain. Our previous study has established several gene vectors and transfection systems for stem cells, on which this project is based. We will try to modify stem cells by two different methods while adopting the concept of Targeted Drug Delivery System (TDDS). Cell surface coating is one of our ideas. MSCs will be coated with targeted peptide either by a transient coating method using palmatic acid-peptide or by biotin-streptavidin linkage. Another trying is to transfect MSCs with CXCR4 gene using non-viral vector combination with reverse transfection system, in order to induce active targeting of MSCs. Upon the research of MSCs targeting, further investigation will focus on the gene engineered MSCs with brain-derived neurotrophic factor (BDNF), for the sake of building a new compound functional targeted vehicle which could carry therapeutic genes targeting to brain lesions as well as differentiation into neural cells by itself. Construction of this new multi-functional targeted vehicle based on stem cells will expand the research area of TDDS, which has scientific significance and shows innovation. This new vehicle has promising prospect and it will promote the development of targeted therapy of stem cells with the joint of many subjects.

骨髓间充质干细胞(MSCs)由于在特定的条件下可分化为神经细胞，在脑部疾病的治疗中被认为具有巨大的潜力。缺血性脑卒中占到全部脑血管病的约80%，成为人类死亡的杀手。研究显示缺血性脑卒中引起的血脑屏障的某些变化为MSCs向脑的分布提供了前提。本项目将在课题组干细胞基因载体研究的基础上，尝试以靶向药物转运体系（TDDS）的理念，通过PA-peptide嵌合/生物素-链霉亲和素桥梁连接特异性靶向肽于干细胞表面，或采用非病毒载体结合反向转染体系高效导入CXCR4基因，介导干细胞的主动靶向。进一步通过脑源性神经营养因子基因重组，构建能趋向脑病变部位、携载治疗基因、同时载体自身又能向神经细胞分化，利于神经修复的全新复合功能靶向载体用于缺血性脑卒中的治疗。基于干细胞的新型复合生物载体的构建，将扩展TDDS的研究领域，具有重要的科学意义和创新性，也将促进学科交叉及干细胞靶向治疗的发展，具有很好的应用前景。

以靶向药物转运体系（TDDS）的理念，将特异性针对脑部病变组织的靶向肽通过适当的修饰使其有效连接到骨髓间充质干细胞（mesenchymal stem cells, MSCs）表面，介导干细胞的主动靶向，同时采用非病毒载体高效安全地导入治疗基因，构建全新的能趋向脑部疾病、携载基因而自身又具有神经生长治疗作用的复合功能生物载体，并结合安全性评价等，全面评估MSCs作为脑靶向生物载体的可行性及其针对缺血性脑卒中的治疗前景。.通过本项目研究成功构建复合功能细胞基因载体Pep-MSCs，流式细胞技术测定靶向肽有较高的修饰效率，激光共聚焦显微镜观察表明靶向肽成功地修饰到了细胞膜表面，MTT检测和MSCs神经分化功能评价均表明此修饰方法安全有效。在体外OGD模型中，与未经修饰的MSCs相比，Pep-MSCs显示出更高的迁移活性，同时靶向肽的修饰并未影响MSCs对于受损神经细胞的保护作用。将构建的Pep-MSCs尾静脉移植到MCAO大鼠体内后，小动物活体成像以及组织切片结果显示，靶向肽能介导MSCs到达缺血病灶，增强MSCs的归巢性，同时减少MSCs在非病灶组织的分布。另一方面，利用已经建立的非病毒转染体系对MSCs进行基因改造，非病毒载体SP能高效安全地转染miR-133b，并且转染miR-133b后的MSCs对受损神经细胞显示更好的保护作用。进一步地，结合基因转染和靶向肽修饰，携载miR-133b的Pep-MSCs在尾静脉移植到MCAO大鼠体内后显示了较好的治疗效果，mNSS评分显示miR-pep-MSCs有更好的神经保护的作用，组织切片结果表明基因转染和靶向肽修饰都未影响MSCs的神经分化功能。.本项目成功构建Pep-MSCs，其对缺血性脑卒中病灶组织有很强的靶向性，作为复合功能基因靶向载体，其能携载治疗基因miR-133b到达病灶组织，又能发挥MSCs自身的治疗作用，在缺血性脑卒中的治疗中显示了很好的应用潜力。通过本项目研究已发表论文13篇，获得授权发明专利2项，培养研究生2名，并获得2016国家自然科学基金国际合作重点项目（81620108028 基于干细胞的新型基因靶向传递系统若干关键问题研究）的资助。