靶向微泡抑制炎症改善微环境对MSC治疗缺血性胆道病变的促进作用及其机制

The ischemic-type biliary lesions (ITBLs) are regarded as the major complication in patients with orthotopic liver transplantation (OLT). Our previous research project has verified contrast-enhanced ultrasonography (CEUS) as the solution to the difficulty of early diagnosis of ITBLs, but the lack of efficient therapeutic approach is another bottleneck which needs further research. Although an attempt on mesenchymal stem cells (MSCs) has obtained some encouraging results in animal experiments, unsatisfied results have been showed on ITBLs patients. The excessive inflammatory response in microenvironment adverse to the MSCs survival probably attributes to the block of its curative effect. Our current project attempts to investigate the feasibility and mechanism of targeted anti-inflammation therapy on improvement of the microenvironment of the ischemic area, MSCs survival and its efficiency to improve therapeutic efficacy of MSCs in animal experiments. The diacylglycerol kinase (DGK) gene to T cell, which is the key molecule to inhibit excessive inflammatory response of ITBLs, is uploaded with ultrasound targeted microbubble (UT-MBs) modified by CD3 antibody. These UT-MBs bind T cell specifically then the DGK gene is targeted released to T cell, to up-regulate the expression of DGKs and induce immunological tolerance of T cell. The targeted anti-inflammation therapy may not only inhibit inflammation and improve microenvironment of MSCs for promotion of its survival and efficiency, but also provide a novel collaborative treatment system in application of MSC therapy besides ITBLs. The results of the project are expected to have important implications for preferable therapeutic effect and prognosis of ITBLs and OLT.

缺血性胆道病变（ITBL）是肝移植最严重的并发症，其早诊难题在课题组前期研究中已基本解决，缺乏有效治疗手段成为瓶颈。间质干细胞（MSC）在动物实验中初显疗效，但在人的尝试性应用却未成功。微环境中过于强烈的炎症反应不利于MSC存活可能是阻碍其疗效发挥的重要原因。本项目拟利用兔模型探讨通过靶向抗炎治疗来改善缺血区微环境、促进MSC存活及生物效应发挥、从而提高ITBL疗效的可行性及其机制。利用超声微泡载体，负载针对ITBL过度炎症反应的关键分子- - T细胞DGKs（甘油二酯酶）基因，以T细胞特异性抗体CD3修饰，实现对T细胞的主动靶向富集，并智能控释DGKs基因，上调DGKs表达、诱导T细胞免疫耐受，实现抑制炎症、改善微环境、促进MSC疗效的目的。本项目可能为包括ITBL在内的众多MSC治疗领域提供一种可促进其长期疗效的新型靶向协同治疗体系，预期结果对提高ITBL疗效、改善肝移植预后将有重要意义

缺血性胆道病变（ITBL）是肝移植最严重的并发症，其早诊难题在课题组前期研究中已基本解决，缺乏有效治疗手段成为瓶颈。间质干细胞（MSC）在动物实验中初显疗效，但在人的尝试性应用却未成功。MSC向靶组织归巢差可能是阻碍其疗效发挥的重要原因。本项目利用超声微泡联合超声辐照过表达MSC表面特异性趋化因子受体，探讨通过改善MSC归巢及生物效应发挥、从而提高ITBL疗效的可行性及其机制。主要研究内容及研究结果如下：1）鉴于DNA存在生物安全性问题，课题组首先确定及制备所用的基因物质。经过基因重组技术构建携带报告基因或目的基因的重组质粒，成功制备出翻译能力强和稳定性高的eGFP-mRNA及CXCR5-mRNA；2）获得负电荷微泡。成功制备出粒径分布均一和稳定性好的负电荷微泡；3）探讨超声靶向协同体系能否成功改造MSC，从而为改善ITBL疗效奠定基础。结果证实负电荷微泡联合超声辐照能成功将mRNA递送至骨髓间充质干细胞内；4）探讨超声靶向协同体系改造MSC的安全性，结果证实负电荷微泡联合超声辐照介导mRNA递送至MSCs内具有良好的安全性；5）探讨mRNA改造MSC治疗炎症性疾病鼠模型的有效性，结果证实mRNA改造的MSC在治疗炎症性疾病具有很好的疗效。本项目可能为包括ITBL在内的众多MSC治疗领域提供一种可促进其长期疗效的新型协同治疗体系，预期结果对提高ITBL疗效、改善肝移植预后将有重要意义。