腺病毒转染铁蛋白质粒作为报告基因监测间充质干细胞移植的磁共振研究

It will be of great value to develop gene-based endogenous magnetic resonance imaging (MRI) contrast using gene transfection methods and using MRI to mointor the distrubution and migration of stem cells after transplantation. Whether the frequently used adeno-associated viral vectors (AVVs) and ferritin have any influence on the features of stem cell and the safty concerned with transplantation. Forthermore, the relationship between ferritin synthetized and MRI signal intensity as well as whether ferritin can serve as a stable and long-termed MRI contrast is still undefined. In this delciate designed work, we scheme to utilize the adeno-associated viral vector to transfer ferritin plasmid into bone marrow-derived mesenchymal stem cells, and using 7 Telsa MRI device to analyze the signal variation before stem cell transplantation in vitro and after transplantation in vivo. Combined with histopathogological, molecular biological and biochemistrical indicators, we will dynamically monitor and evaluate the effects of AVVs transfection and ferritin to the stem cell molecular phenotype, proliferation, differentation and apoptosis properties and its evolution mechanisms for a long period of time, and exploring the influence of AVVs and ferritin to the internal environment, inflammatory factors, glycoprotein alteration, macrophage reaction and its relevant menchanisms. The security efficiency, stabilization, accuracy and reliability of AVVs and ferritin as endogenous MR contrast will be dynamically quantitative analyzed. In general, this work will provide profound technical reference and theoretical foundation for stem cell therapeutic effects and surveillance after transplantation.

用基因转染法使干细胞合成铁蛋白作为内源性示踪剂并利用磁共振监测干细胞移植后分布迁移可望解决目前无长期、定量有效监测干细胞的技术。但目前常用的腺病毒载体及铁蛋白合成是否会影响干细胞特性及对干细胞移植安全性的影响尚不明确，铁蛋白合成量与磁共振信号之间的量效关系及铁蛋白能否作为稳定的示踪剂长期有效准确监测干细胞等重要问题尚待明确。本研究用腺病毒转染铁蛋白质粒进入间充质干细胞，利用7T小动物磁共振仪监测离体及在体移植后干细胞内铁蛋白信号变化，佐以组织病理学、分子生物学等指标，长时间动态量化分析腺病毒转染铁蛋白质粒对干细胞分子表型及免疫特征、增殖、分化、凋亡等生物学行为影响及机制，探索其对在体移植后干细胞所处内环境影响、炎性因子、糖蛋白及巨噬细胞反应、变化趋势及相关机制,长期动态量化分析腺病毒质粒转染铁蛋白作为内源性示踪剂的安全性、稳定性、准确性及可靠性，为干细胞移植后监测及评估提供可靠技术及理论。

用腺病毒转染铁蛋白质粒的方法使干细胞合成铁蛋白作为内源性示踪剂，利用磁共振监测转染了铁蛋白质粒的干细胞分布迁移可望解决目前无长期、定量有效监测干细胞的技术。但目前常用的腺病毒载体及铁蛋白合成是否会影响干细胞特性及对干细胞移植安全性的影响尚不明确，铁蛋白合成量与磁共振信号之间的量效关系及铁蛋白能否作为稳定的示踪剂长期有效准确监测干细胞等重要问题尚待明确。本研究用腺病毒转染含铁蛋白重链基因与荧光基因的双基因质粒进入间充质干细胞，用MTT比色法佐以组织病理学、分子生物学等指标，长时间动态量化分析腺病毒转染铁蛋白质粒对干细胞分子表型及免疫特征、增殖、分化、凋亡等生物学行为影响，发现腺病毒转染铁蛋白质粒作为内源性示踪剂具有较好的安全性。利用7T小动物磁共振仪与3T磁共振仪监测离体干细胞内铁蛋白信号变化，佐以光学显微镜、分子生物学定量检测细胞内铁蛋白含量，发现铁蛋白合成量与磁共振信号之间具有较高的量效关系，特别是T2mapping与eswan技术能量化磁共振信号，在长期动态量化分析腺病毒质粒转染铁蛋白作为内源性示踪剂在体外具有较好的稳定性、准确性及可靠性，为干细胞移植后监测及评估提供可靠技术及理论，同时，通过初步临床应用研究表明，该技术还能无损伤性定量监测帕金森氏病、糖尿病等有关代谢性疾病。