磁共振示踪小鼠树突状细胞归巢淋巴结的初步研究
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摘要
目的:尝试采用磁共振成像(magnetic resonance imaging,MRI)监测磁标记树突状细胞(dendritic cell,DC)归巢引流淋巴结。方法:DC由小鼠骨髓细胞经诱导分化获得。探针为烷基化低分子量聚乙烯亚胺(PEI2k)包裹的超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)纳米颗粒,标记方法为成熟DC与探针共孵育过夜。收集细胞,行标记效果、细胞分子表型分析。将标记的成熟DC注入小鼠足底,行不同时间点MRI,观察DC归巢淋巴结,72 h后,取腘窝淋巴结送病理检查。结果:DC纯度高达90%。普鲁士兰染色和激光共聚焦成像显示探针位于细胞内。流式细胞术(fluorescence activated cell sorting,FACS)分析表明,标记过程对DC的成熟表型无明显影响。体内MRI和淋巴结病理显示,24 h后标记DC在淋巴结内聚集。结论:采用N-alkylPEI2k/SPIO纳米探针标记DC后,MRI可以清楚地监测其在体内归巢淋巴结的情况。
Objective:To track homing of magnetically-laden dendritic cells(DCs)into the draining lymph nodes using magnetic resonance(MR)imaging. Methods:Mature DCs generated from murine bone marrow cells were labeled with a laboratorially-synthesized alkylated-polyethyleneimine(PEI2k)coated superparamagnetic ironoxide(SPIO)nanoparticle. Perl's staining,confocal laser scanning microscopy(CLSM),and fluorescence activated cell sorting(FACS)were followed to analyze labeling results and expression of mature phenotype,respectively. For in vivo MR imaging,gradient amount of DCs were subcutaneously injected into the footpads of three mice.MR scanning was performed at different time points. At 72 h after injection,mice were sacrificed to extract the popliteal lymph nodes for pathological examination. Results:A final purity of about 90% DC was achieved by this culture method. Probes located in cytoplasm after overnight labeling,as evidenced by Perl's staining and CLSM. Labeling process did not impact obviously on the maturation phenotypes of DCs. Both MRI and pathological studies documented the successful migration of labeled DCs into popliteal nodes.Conclusion:It is feasible to utilize MR imaging to track the in vivo migration of DCs labeled with N-alkyl-PEI2k/SPIO nanoprobe.
Objective:To track homing of magnetically-laden dendritic cells(DCs)into the draining lymph nodes using magnetic resonance(MR)imaging. Methods:Mature DCs generated from murine bone marrow cells were labeled with a laboratorially-synthesized alkylated-polyethyleneimine(PEI2k)coated superparamagnetic ironoxide(SPIO)nanoparticle. Perl's staining,confocal laser scanning microscopy(CLSM),and fluorescence activated cell sorting(FACS)were followed to analyze labeling results and expression of mature phenotype,respectively. For in vivo MR imaging,gradient amount of DCs were subcutaneously injected into the footpads of three mice.MR scanning was performed at different time points. At 72 h after injection,mice were sacrificed to extract the popliteal lymph nodes for pathological examination. Results:A final purity of about 90% DC was achieved by this culture method. Probes located in cytoplasm after overnight labeling,as evidenced by Perl's staining and CLSM. Labeling process did not impact obviously on the maturation phenotypes of DCs. Both MRI and pathological studies documented the successful migration of labeled DCs into popliteal nodes.Conclusion:It is feasible to utilize MR imaging to track the in vivo migration of DCs labeled with N-alkyl-PEI2k/SPIO nanoprobe.
引文
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[2]曹新山,姜兴岳,毛锡金,等.DWIBS评价SPIO标记树突状细胞治疗兔VX2肝癌的疗效[J].医学影像学杂志,2012,22(9):1550-1553.
[3]林嘉盈,李晓燕.治疗头颈部肿瘤的树突状细胞疫苗的研究进展[J].医学综述,2010,16(22):3393-3396.
[4]de Vries IJ,Lesterhuis WJ,Barentsz JO,et al.Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy[J].Nat Biotechnol,2005,23(11):1407-1413.
[5]Liu G,Xie J,Zhang F,et al.N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient si RNA delivery[J].Small,2011,7(19):2742-2749.
[6]Sun S,Zeng H,Robinson DB,et al.Monodisperse MFe2O4(M=Fe,Co,Mn)Nanoparticles[J].J Am Chem Soc,2004,126(1):273e9.
[7]吴昆,拜合提亚·阿扎提,王文光,等.小鼠骨髓来源树突状细胞培养鉴定和诱导T淋巴细胞增殖[J].南京医科大学学报(自然科学版),2011,31(8):1128-1133.
[8]Kobukai S,Baheza R,Cobb JG,et al.Magnetic nanoparticles for imaging dendritic cells[J].Magn Reson Med,2010,63(5):1383-1390.
[9]Verdijk P,Scheenen TW,Lesterhuis WJ,et al.Sensitivity of magnetic resonance imaging of dendritic cells for in vivo tracking of cellular cancer vaccines[J].Int J Cancer,2007,120(5):978-984.
[10]Mou Y,Hou Y,Chen B,et al.In vivo migration of dendritic cells labeled with synthetic superparamagnetic iron oxide[J].Int J Nanomedicine,2011(6):2633-2640.
[11]Rohani R,de Chickera SN,Willert C,et al.In vivo cellular MRI of dendritic cell migration using micrometer-sized iron oxide(MPIO)particles[J].Mol Imaging Biol,2011,13(4):679-694.