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SD Rat Adipose Derived Stem Cells Labeled with Ultrasmall Superparamagnetic Iron Oxide
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摘要:
目的 评估超小超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide, USPIO)标记SD大鼠脂肪来源干细胞(adipose derived stem cells, ADSCs)的有效性及安全性, 探讨标记细胞体外磁共振成像(magnetic resonance imaging, MRI)特点。 方法 将USPIO 40 μg/ml及多聚赖氨酸(poly-L-lysine, PLL)1.5 μg/ml的培养基与ADSCs共孵育培养24 h, 检测USPIO标记的有效性及安全性, 并用MRI对标记细胞进行体外成像。 结果 普鲁士蓝染色显示USPIO标记ADSCs的阳性率为99%, 透射电镜提示USPIO颗粒主要位于胞质内溶酶体中; 台盼蓝染色实验显示活细胞数大于95%, MTS[3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, 溴化噻唑蓝四氮唑]实验提示USPIO浓度为10、20、40、80和160 μg/ml时不影响ADSCs增殖。ELISA结果表明标记细胞与未标记细胞组间培养液中血管内皮生长因子(vascular endothelial growth factor, VEGF)水平无显著差异; 体外条件下, MRI图像信号强度与标记细胞数量呈正相关。 结论 USPIO标记ADSCs安全有效, MRI图像信号强度与标记细胞数量存在一定相关性, 提示USPIO可用于在体条件下MRI成像示踪标记细胞。 Abstract:Objective To investigate the efficacy and safety of ultrasmall superparamagnetic iron oxide (USPIO) labeling adipose derived stem cells (ADSCs) of SD rats and explore the feasibility of tracing labeled cells with magnetic resonance imaging (MRI). Methods ADSCs were incubated with culture medium containing USPIO and poly-L-lysine (PLL) for 24h. The efficacy and safety of labling with USPIO was assessed, and the labeled cells were imaged with MRI in vitro. Results Prussian blue staining showed the percentage of labeled ADSCs reached 99% after coincubating for 24 h. Transmission electron microscopy showed iron particles inside the cells were mainly in lysosomes. Trypan-Blue stain showed the proportion of living cells was greater than 95%. MTS[3-(4, 5-dimethylthiazol-2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium] experiments suggested that USPIO at different concentrations (10, 20, 40, 80, 160μg/ml) exerted no significant influence on the proliferation of ADSCs. Enzyme-linked immunosorbent assay (ELISA) revealed VEGF level was not significantly different between labeled cells and unlabeled cells. The signal intensity of MRI was positively correlated with the amount of labeled cells in vitro. Conclusion USPIO is safe and efficient in labeling ADSCs and the signal intensity of MRI is associated with the amount of labeled cells, indicating that USPIO can be used for tracing the labled cells under MRI in vitro. -
图 1 超小超顺磁性氧化铁标记脂肪来源干细胞普鲁士蓝染色及透射电镜观察
A.普鲁士蓝染色(蓝色为铁颗粒)，标记阳性率接近100%，蓝染颗粒多位于细胞核周围(× 400); B.核固红复染(红色为胞核和部分胞质); C.透射电镜示细胞形态完整，胞质中散在黑色USPIO颗粒(×6000); D.透射电镜示黑色USPIO颗粒位于溶酶体中，细胞膜凹陷吞噬黑色颗粒(箭头) (×25 000)
USPIO:同表 1
图 2 不同数量级超小超顺磁性氧化铁标记脂肪来源干细胞体外磁共振成像
1→6: USPIO标记ADSCs细胞数递增(ml-1) 5 ×102、5 ×103、5 ×104、1 ×105、2. 5 ×105、5 ×105; A.琼脂糖24孔板T1序列成像，除6号孔信号出现信号减低外，其余各细胞孔与空白对照孔无明显差异; B.琼脂糖24孔板T2序列成像，随细胞数增多，孔板信号强度逐渐减低; C.琼脂糖24孔板T2*序列成像，T2*图像上可见磁敏感伪影出现
USPIO:同表 1; ADSCs:脂肪来源干细胞
表 1 脂肪来源干细胞培养液血管内皮生长因子含量
时间(h) VEGF (pg/ml，x ± s) P值 未标记干细胞培养液 USPIO标记干细胞培养液 6 22. 63 ± 6. 39 16. 76 ± 4. 82 0. 75 12 38. 47 ± 7. 32 36. 77 ± 7. 60 0. 18 24 52. 69 ± 6. 54 55. 39 ± 7. 06 0. 76 48 79. 47 ± 7. 73 87. 91 ± 6. 73 0. 20 P值 ＜0. 01 ＜0. 01 VEGF:血管内皮生长因子; USPIO:超小超顺磁性氧化铁 
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表 2 各数量级超小超顺磁性氧化铁标记脂肪来源干细胞的T2及T2*值
孔编号 细胞数量(ml －1) T2 (ms) T2* (ms) 1 5 × 102 104. 08 ± 5. 16 39. 38 ± 15. 63 2 5 × 103 94. 52 ± 4. 82 24. 97 ± 9. 16 3 5 × 104 75. 13 ± 3. 86 13. 37 ± 6. 25 4 1 × 105 61. 15 ± 2. 78 6. 20 ± 5. 00 5 2. 5 × 105 3. 73 ± 11. 57 0 6 5 × 105 0 0 空白 0 107. 65 ± 4. 48 40. 57 ± 14. 32
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[1] Wei HM, Wong P, Hsu LF, et al. Human bone marrow-derived adult stem cells for post-myocardial infarction cardiac repair:current status and future directions[J]. Singapore Med J, 2009, 50:935-942. http://www.ncbi.nlm.nih.gov/pubmed/19907881 [2] Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium[J]. Nature, 2001, 410:701-705. doi: 10.1038/35070587 [3] Aassmus B, Rolf A, Erbs S, et al. Clinical outcome 2 years after intracornoary administration of bone marrow-derived progenitor cells in acute myocardial infarction[J]. Circ Heart Fail, 2010, 3:89-96. doi: 10.1161/CIRCHEARTFAILURE.108.843243 [4] Kirchin MA, Runge VM. Contrast agents for magnetic resonance imaging:safety update[J]. Top Magn Reson Imaging, 2003, 14:426-435. doi: 10.1097/00002142-200310000-00007 [5] Shapiro EM, Skrtic S, Koretsky AP. Sizing it up:cellular MRI using micron-sized iron oxide particles[J]. Magn Reson Med, 2005, 53:329-338. doi: 10.1002/mrm.20342 [6] Sun R, Dittrich J, Le-Huu M, et al. Physical and biological characterization of superpara-magnetic iron oxide-and ultrasmall superparamagnetic iron oxide-labeled cells:a comparison[J]. Invest Radiol, 2005, 40:504-513. doi: 10.1097/01.rli.0000162925.26703.3a [7] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines[J]. Physiol Rev, 2003, 83:835-870. doi: 10.1152/physrev.2003.83.3.835 [8] Takahashi M, Li TS, Suzuki R, et al. Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury[J]. Am J Physiol Heart Circ Physiol. 2006, 291:H886-893. doi: 10.1152/ajpheart.00142.2006 [9] Lau JF, Anderson SA, Adler E. Imaging approaches for the study of cell-based cardiac therapies[J]. Nat Rev Cardiol, 2010, 7:97-105. doi: 10.1038/nrcardio.2009.227 [10] Fahlvik AK, Klaveness J, Stark DD. Iron oxides as MR imaging contrast agents[J]. J Magn Reson Imaging, 1993, 3:187-194. doi: 10.1002/jmri.1880030131 [11] McNeill A, Birchall D, Hayflick SJ, et al. T2* and FSE MRI distinguishes four subtypes of neurodegeneration with brain iron accumulation[J]. Neurology, 2008, 70:1614-1619. doi: 10.1212/01.wnl.0000310985.40011.d6 [12] Yanagawa Y, Tsushima, Y, Tokumaru A, et al. A Quantitative Analysis of Head Injury Using T2* -Weighted Gradient-Echo Imaging[J]. J Trauma, 2000, 49:272-277. doi: 10.1097/00005373-200008000-00013 [13] McCarville MB, Hillenbrand CM, Loeffler RB, et al. Comparison of whole liver and small region-of-interest measurements of MRI liver R2* in children with iron overload[J]. Pediatr Radiol, 2010, 40:1360-1367. doi: 10.1007/s00247-010-1596-8 [14] Wang ZJ, Fischer R, Chu Z, et al. Assessment of cardiac iron by MRI susceptometry and R2* in patients with thalassemia[J]. Magn Reson Imaging, 2010, 28:363-371. doi: 10.1016/j.mri.2009.12.001 [15] Zhang MW, Huan Y, Xu J, et al. In vitro MR imaging of superparamagnetic iron oxide labeled mesenchymal stem ceils[J]. Chin J Med Imaging Technol, 2006, 22:1129-1134. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zhsywk201711018 [16] Hill JM, Dick AJ, Raman VK, et al. SeriaI cardiac magnetic resonance Imaging of injected mesenchymal stem cells[J]. Circulation, 2003, 108:1009-1014. doi: 10.1161/01.CIR.0000084537.66419.7A [17] Foster-Gareau P, Heyn C, Alejski A, et al. Imaging single mammalian cells with a 1.5 T clinical MRI scanner[J]. Magn Reson Med. 2003, 49:968-971. doi: 10.1002/mrm.10417 [18] Rad AM, Arbab AS, Iskander AS, et al. Quantification of superparamagnetic iron oxide (SPIO) -labeled cells using MRI[J]. J Magn Reson Imaging, 2007, 26:366-374. doi: 10.1002/jmri.20978 -
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