间充质干细胞多模态示踪方法的应用

Application in multimodality tracking of mesenchymal stem cells

目的合成具备MR显像和基因传输功能的纳米载体,探讨载体对间充质干细胞(MSCs)的基因传输功能及其联合生物发光成像(BLI)和MRI对MSCs双模态示踪的能力。方法合成三元共聚物聚乙二醇-聚天冬氨酸(聚乙烯亚胺)-超顺磁性氧化铁纳米颗粒载体(PAI/SPION);利用凝胶阻滞实验分析载体携带质粒(pDNA)的能力;电位及粒度测定仪测量载体复合pDNA后的粒径和电位;采用大鼠股骨骨髓分离培养MSCs;采用流式细胞仪、激光共聚焦显微镜评估PAI/SPION/pDNA对MSCs的基因转染效率;联合BLI和MRI双模态示踪MSCs。结果成功合成了载体PAI/SPION。氨基与质粒磷酸根的摩尔比(N/P)=3.0时,PAI/SPION可完全复合pDNA。N/P=12时,粒径趋于稳定,为(74.8±8.1)nm,电位为(12.2±1.5)mV,载体对MSCs的基因转染率为(71.2±2.3)%。激光共聚焦显微镜下,胞浆内可见大量表征PAI/SPION的绿色荧光和表征pDNA的红色荧光,且MSCs生物发光强度最高,T2*WI标准化信号强度最低。结论本研究成功合成了MRI可视的基因传输载体PAI/SPION;载体可高效传输pDNA至大鼠MSCs,且可成功联合BLI和MRI双模态示踪MSCs。

Objective To synthesize MRI-visible gene transfer nanocarrier and to assess the gene delivery ability of nanocarrier to mesenchymal stem cells（MSCs）and monitored the MSCs using bioluminescence imaging（BLI）and MRI.Methods The terpolymer nanocarrier poly（ethylene glycol）-block-poly（L-aspartic acid）-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles（PAI/SPION）was synthesized.The degree of binding between PAI/SPION and pDNA was determined with agarose gel electrophoresis.The Zeta potential and particle size of polymer vectors were measured with a Zeta-Plus instrument.MSCs were isolated and cultured from the femurs of the rats.The gene transfection efficiency of PAI/SPION/pDNA to MSCs was evaluated by flow cytometry and laser confocal microcopy.And the BLI and MRI were combined to monitor MSCs.Results PAI/SPION vectors were synthesized successfully.PAI/SPION could condense pDNA entirely at a nitrogen of non-viral vector/phosphorus of plasmid（N/P）ratio of 3.0.The particle size of PAI/SPION/pDNA attained a constant size of（74.8±8.1）nm at a N/P ratio of 12,and the Zeta potential reached（12.2±1.5）mV,the transfection efficiency of PAI/SPION/pDNA to MSCs was（71.2±2.3）%.The MSCs showed significantly stronger PAI/SPION（green）and pDNA（red）fluorescence under the laser confocal microcopy,the bioluminescence light intensity of the MSCs attained the highest values and the normalized MR T2＊WI signal intensity attained the lowest values.ConclusionThe study successfully synthesizes an MRI-visible non-viral gene vector PAI/SPION,which possesses a higher transfection efficiency in transferring pDNA into MSCs and can dually monitor the MSCs using BLI and MRI.