针对缺血性疾病利用干细胞膜微粒在细胞间信号传递的载体功能研究

Mesenchymal stem cells (MSC) and gene technology to treat ischemic disease is effective, but the mechanism and application are still in dispute.Our study shows that: MSC apoptosis induced by special condition can release a large number of microparticles (MP), which will not only promote the vascular endothelial cells to proliferate, migratie and form tube formation, but also promote angiogenesis in ischemic limbs to be newborn. It's confirmed the MP can carry the protein and the genetic information to the target cells, which indicates that the protective effection of MSC can be transfered to the target cell by the MP, and the information of MSC acts on target cells. By this way, it is expected that MSC can release more MP to promote target cells to creat special biological effects, which carried the siRNA, proteins and cytokines accordding to our plan. So it may lead a possibility that the gene transfection technology can be promoted using in human tissue cells. Taking ischemic disease as research objects,we modifyed MSC by gene technology with siRNA of IL-6 and HGF plasmid to acquire MP,and transfect ischemic cell. the gene and related protein expressing, function will be detected on every step by PCR, Western blot and ELISA, investigating the effects of the specified cell factor and siRNA passed by MP. A possible systerm which can optimize MP is expected, in order to provide a new therapy approach for ischemic disease.

间充质干细胞（MSC）及基因技术治疗缺血性疾病有效，而机制与应用方式存在争议。我们前期研究表明：诱导MSC凋亡可释放大量膜微粒（MP），MP不但促进内皮细胞增殖、迁移和管状结构形成，也能促进缺血肢体血管新生。并且证实了MP将其携带的蛋白质及遗传信息转移至靶细胞，证明MSC的促生物效应可通过MP传递给靶细胞。期望利用这一原理将预期的siRNA、细胞信号及细胞因子传递给靶细胞，将MP作为细胞信号传递的载体，有利于基因技术在人体组织细胞上的应用。以大鼠心梗模型为研究对象，用基因技术处理MSC，使其高表达有益（HGF）或低表达有害（IL6）效应分子，然后诱导产生MP、作用靶细胞，通过荧光定量PCR、Western Blot及ELISA检测基因序列及其表达蛋白在MP与靶细胞水平的变化。观察细胞因子及siRNA通过MP传递的效果，期望建立一种优化MP的方法，为缺血性疾病治疗提供一种新的方法。

基于前期对间充质干细胞来源的膜微囊在新生血管作用的研究基础。膜微囊被认为是细胞信息传递的重要介质。了解膜微囊作用的机制是调控膜微囊作用靶细胞的关键。我们通过共聚焦与流式细胞术证实膜微囊对靶细胞的作用主要通过内化，并用生物学方法对其内化的机制进行进一步的研究。膜微囊根据源细胞的特性所处微环境的不同，可携带不同的物质对靶细胞进行生理或病理性调控。以临床缺血性疾病患者循环血中膜微囊为研究对象，流式细胞术对临床循环血中膜微囊来源特性进行了研究，iTARQ技术对其携带蛋白质进行GO及Pathway分析，对膜微囊在缺血心疾病患者疾病发展中作用有了一定提示，循环血中膜微囊有可能作为缺血性疾病的检测标志，另外在外科冠脉搭桥手术的患者可能成为患者术后预后的独立预测因子。