纳米-脐带间充质干细胞治疗小鼠缺血性卒中的研究

Ischemic stroke is one of the leading causes of morbidity and mortality in China, and no approved treatment is available except thrombolysis, which has a short time window. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are able to improve the outcomes of ischemic stroke, partially through inducing host brain plasticity, angiogenesis and neurogenesis, and secreting the neurotropic factors, but the deficiency of hUC-MSCs limits its clinical application. Emerging evidence has shown that carbon nanomaterials, with satisfactory physical and chemical functions and high biocompatibility, are regarded promising for the neuronal tissue-engineering. Implantation of the scaffolds composed of carbon nanotube (CNT) and neural stem cells (NSC) to the damaged brain tissue after ischemic stroke can promote the adhesion, growth, proliferation and differentiation of NSC, which contributes to the improved prognosis. Grapheme and carbon nanocage (CNC), which promises marked advances to CNT as the scaffolds, has been rarely studied. Our preliminary studies have shown that CNC can promote the growth and proliferation of hUC-MSCs. In this study, we will investigate the bioactivity of grapheme/CNC as the scaffolds to make the more compatible nanomaterials with N-doping technology based on the studies of CNT. In addition, we will implant the scaffolds composed of grapheme/CNC and hUC-MSCs into the ischemic brain areas to evaluate the therapeutic effects and to explore the underlying mechanism, which will provide evidence for the clinical translation of hUC-MSCs- carbon nanomaterials therapy for ischemic stroke.

缺血性卒中在我国是高致死率、致残率的疾病，人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）已被证实能够改善卒中预后。但其来源的不足大大限制了临床应用，细胞生物工程的发展为这一问题的解决提供了新的思路。碳纳米材料是神经组织工程领域中的新兴材料之一。其中，碳纳米管（CNT）已被证实能与神经干细胞组成细胞生物支架，促进干细胞的粘附生长及增殖分化，改善卒中预后。而更为新兴的石墨烯和碳纳米笼，相较于CNTs具有作为生物支架更优越的性能。我们前期结果显示碳纳米笼能促进hUC-MSCs的生长增殖。本课题将进一步探索石墨烯和碳纳米笼作为生物支架的性能，并引入氮掺杂技术以期筛选出具有更佳生物相容性的材料，将hUC-MSCs种于其上并植入卒中动物模型，评估其疗效及其具体机制，为干细胞治疗缺血性卒中临床转化奠定理论基础。

缺血性卒中在我国是高致死率、致残率的疾病，人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）已被证实能够改善卒中预后。但其来源的不足大大限制了临床应用，细胞生物工程的发展为这一问题的解决提供了新的思路。碳纳米材料是神经组织工程领域中的新兴材料之一。其中，碳纳米管（CNT）已被证实能与神经干细胞组成细胞生物支架，促进干细胞的粘附生长及增殖分化，改善卒中预后。而更为新兴的石墨烯（graphene）和碳纳米笼（CNCs），相较于CNTs具有作为生物支架更优越的性能。本课题在前期研究基础上，进一步探索石墨烯和碳纳米笼作为生物支架的性能；比较不同碳纳米材料对hUC-MSCs生长、增殖、分化的影响；将碳纳米材料-hUC-MSCs植入缺血性卒中动物模型，评估其疗效及其具体机制。.结果发现：hUC-MSCs体外培养成纤维样细胞形态，螺漩成簇生长，流式细胞检测结果提示间充质干细胞表面标志物CD73、CD90、CD105，不表达造血干细胞表面标志物HLA-DR, CD45, CD34, CD14；免疫荧光染色（MAB1287）结果显示，生长于石墨烯与碳纳米笼的hUC-MSCs较对照组粘附、生长增殖能力更强；MTT结果提示石墨烯、碳纳米笼均能够增强体外培养的hUC-MSCs的细胞存活率；使用MAP-2作为神经元特异性的标志物，免疫荧光结果显示hUC-MSCs与石墨烯及碳纳米笼共培养后，均能够检测到MAP-2阳性细胞，而在对照组中则较少有hUC-MSCs分化为神经元；ELISA方法测定NGF、VEGF表达水平，结果显示碳纳米笼显著增强了hUC-MSCs表达NGF、VEGF能力；神经功能评分、TTC染色以及脑组织水肿程度检测结果发现与CNCs共培养的hUC-MSCs具有更强的减轻MCAO模型小鼠脑损伤的功能，并能够下调氧化应激指标4-HNE，3-NT水平，以及炎症因子IL-1β、TNF-α、IL-6、iNOS的释放。.结论：通过体内外实验，我们证实石墨烯、碳纳米笼能够提高体外培养人脐带间充质干细胞的存活率、生长增殖及分化能力；碳纳米材料-hUC-MSCs具有减轻缺血性脑损伤功能，且体内实验结果表面其保护作用的机制可能与减轻MCAO后脑组织的氧化应激反应及炎症反应有关。