双标记示踪脂肪干细胞治疗下肢缺血的研究  

Tracking of dual-labeled adipose-derived stem cells in therapying mouse hind-limb ischemia

作　　者：袁福康[1] 陆信武[1] 秦金保[1] 彭智猷[1] 叶开创[1] 杨心蕊[1] 黄丽佳[1] 蒋米尔[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院血管外科上海交通大学血管病诊治中心,200011

出　　处：《中华实验外科杂志》2015年第11期2676-2680,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金项目资助项目（81370423）;上海市科学技术委员会资助项目（134119a2100）

摘　　要：目的 探讨超顺磁氧化纳米铁颗粒（SPIO）标记绿色荧光蛋白（GFP）转基因小鼠来源的脂肪干细胞（GFP-ADSCs）治疗C57小鼠下肢缺血的可行性及效果.方法 取4周龄GFP转基因小鼠的脂肪组织,消化获取GFP-ADSCs,流式检测其表面的干细胞标记.建立C57BL/6左下肢肌肉缺血模型并随机分成3组,每组10只.一组缺血的肌肉组织内注射100μl第3代GFP-ADSCs 1×106个,另一组注射100μl SPIO标记的1×106个GFP-ADSCs,对照组同样部位注射100μl磷酸盐缓冲液（PBS）.3周后通过磁共振成像（MRI）示踪SPIO标记GFP-ADSCs在缺血肌肉内的迁移及转归情况,普鲁士蓝染色检测缺血组织的中SHO标记GFP-ADSCs归巢情况,并通过免疫荧光染色（IF）和免疫组织化学（IHC）行血管性假血友病因子（vWF）、α-平滑肌肌动蛋白（α-SMA）染色以观察缺血肌肉组织内血管新生情况.结果 （1）从GFP转基因小鼠的脂肪组织中可以获得大量GFP-ADSCs,并表达干细胞表面分子标记Sca-1[（80.80±0.23）％]和CD44[（77.50 ±0.16）％];（2）SPIO可以有效地标记GFP-ADSCs,在25 mg/L浓度范围内不影响GFP-ADSCs的增殖;（3）SPIO标记的GFP-ADSCs可以被MRI检测到;（4）3周后SPIO标记的GFP-ADSCs治疗组和GFP-ADSCs治疗组缺血的肌肉组织内可见较多的新生血管,平均血管密度分别为4.39±0.76和4.62 ±0.87,新生血管不仅表达内皮细胞的特异性标记vWF,同时表达GFP,且微血管密度数显著高于PBS组（2.15±0.53,P＜0.05）,但治疗组间差异无统计学意义（P＜0.05）.结论 从GFP转基因小鼠的脂肪组织中可以获取大量的GFP-ADSCs,其能够促进小鼠下肢缺血肌肉组织内的血管新生,SPIO可以有效的标记GFP-ADSCs,且不影响其增殖,其自带的绿色荧光蛋白GFP和SPIO可以同时动态示踪ADSCs在缺血肌肉内的存活、迁移及归巢情况.Objective To investigate the effect and availably of superparamagnetic iron oxide nanoparticle （SPIO）-labeled green fluorescent protein gene （GFP）-Adipose-derived stem cells （ADSCs） treatment in hind-limb ischemia.Methods The surface antigen of GFP-ADSCs were identified by flow cytometry after digesting GFP-ADSCs isolated from the adipose tissues of 4w old GFP mice.Left hind-limb ischemic models of C57BL/6 were established and randomly divided into three groups, ten mice each group.Labeled and without labeled P3 GFP-ADSCs （1 × 10^10/L） were respectively transplanted into the muscle which was hind-limb ischemia of one group C57BL/6 comparing to 100 μl PBS was injected into the same place of the third group.3T magnetic resonance imaging （MRI） was acquired to monitoring the migration and homing of labeled GFP-ADSCs after 3 weeks.The homing of GFP-ADSCs with labeled was tested by Prussian blue staining 3 weeks later, the effect of rebirh vessel in muscle were tested by α-smooth muscle actin （α-SMA） of immunohistochemistry （IHC） and Immunofluorescence （IF） with vascular Von Willebrand factor （vWF）.Results （1） A lot of GFP-ADSCs could be isolated from green fluorescent protein transgenic mice and them also express Sca-1 [（80.80 ±0.23）%] and CD44 [（77.50 ±0.16）%] of stem cell surface antigen;（2） GFP-ADSCs were effectively labeled with SPIO and also proliferated without being affected at the concentration of 25ug/ml;（3） The position where contained SPIO by Prussian blue staining according to the MRI;（4） The α-SMA of IHC indicated that： new blood vessels were distributed among the muscle bundles in treat team after 3 w;Micro vessel density （MVD） ： The new vessel density of labeled and without labeled GFP-ADSCs treat group （4.39 ± 0.76 and 4.62 ± 0.87） were obviously higher than that of Group PBS （2.15 ± 0.53） in the same condition （P 〈 0.05）, while there were no significant difference between two treat group;IF indicated that the n

关 键 词：超顺磁氧化纳米铁颗粒 绿色荧光蛋白 体内示踪 下肢缺血 脂肪干细胞 

分 类 号：R318.04[医药卫生—生物医学工程]