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超小超顺磁性氧化铁体外标记SD大鼠脂肪来源干细胞

曹剑 王怡宁 孔令燕 薛华丹 雷晶 何泳蓝 李琢 孟洁 金征宇

曹剑, 王怡宁, 孔令燕, 薛华丹, 雷晶, 何泳蓝, 李琢, 孟洁, 金征宇. 超小超顺磁性氧化铁体外标记SD大鼠脂肪来源干细胞[J]. 协和医学杂志, 2011, 2(3): 252-257. doi: 10.3969/j.issn.1674-9081.2011.03.013
引用本文: 曹剑, 王怡宁, 孔令燕, 薛华丹, 雷晶, 何泳蓝, 李琢, 孟洁, 金征宇. 超小超顺磁性氧化铁体外标记SD大鼠脂肪来源干细胞[J]. 协和医学杂志, 2011, 2(3): 252-257. doi: 10.3969/j.issn.1674-9081.2011.03.013
Jian CAO, Yi-ning WANG, Ling-yan KONG, Hua-dan XUE, Jing LEI, Yong-lan HE, Zhuo LI, Jie MENG, Zheng-yu JIN. SD Rat Adipose Derived Stem Cells Labeled with Ultrasmall Superparamagnetic Iron Oxide[J]. Medical Journal of Peking Union Medical College Hospital, 2011, 2(3): 252-257. doi: 10.3969/j.issn.1674-9081.2011.03.013
Citation: Jian CAO, Yi-ning WANG, Ling-yan KONG, Hua-dan XUE, Jing LEI, Yong-lan HE, Zhuo LI, Jie MENG, Zheng-yu JIN. SD Rat Adipose Derived Stem Cells Labeled with Ultrasmall Superparamagnetic Iron Oxide[J]. Medical Journal of Peking Union Medical College Hospital, 2011, 2(3): 252-257. doi: 10.3969/j.issn.1674-9081.2011.03.013

超小超顺磁性氧化铁体外标记SD大鼠脂肪来源干细胞

doi: 10.3969/j.issn.1674-9081.2011.03.013
基金项目: 

国家自然科学基金青年科学基金 81000629

详细信息
    通讯作者:

    王怡宁 电话:010-65295509, E-mail:yiningpumc@hotmail.com

  • 中图分类号: R445.2;R35

SD Rat Adipose Derived Stem Cells Labeled with Ultrasmall Superparamagnetic Iron Oxide

More Information
  • 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。
  • 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

    图  3  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数值

    T2及T2*值随USPIO标记ADSCs细胞数量增多而逐渐降低; ΔR2及ΔR2*与USPIO标记ADSCs细胞数呈近线性关系,相关系数r2>0. 9
    USPIO:同表 1; ADSCs:同图 1

    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
    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 <0. 01 <0. 01
    VEGF:血管内皮生长因子; USPIO:超小超顺磁性氧化铁
    下载: 导出CSV

    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*

    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
    下载: 导出CSV
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出版历程
  • 收稿日期:  2011-05-14
  • 刊出日期:  2011-07-30

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