CAJ | 학술논문

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.
목적 탐토초순자양화납미철과립(SPIO)표기록색형광단백(GFP)전기인소서래원적지방간세포(GFP-ADSCs)치료C57소서하지결혈적가행성급효과.방법 취4주령GFP전기인소서적지방조직,소화획취GFP-ADSCs,류식검측기표면적간세포표기.건립C57BL/6좌하지기육결혈모형병수궤분성3조,매조10지.일조결혈적기육조직내주사100μl제3대GFP-ADSCs 1×106개,령일조주사100μl SPIO표기적1×106개GFP-ADSCs,대조조동양부위주사100μl린산염완충액(PBS).3주후통과자공진성상(MRI)시종SPIO표기GFP-ADSCs재결혈기육내적천이급전귀정황,보로사람염색검측결혈조직적중SHO표기GFP-ADSCs귀소정황,병통과면역형광염색(IF)화면역조직화학(IHC)행혈관성가혈우병인자(vWF)、α-평활기기동단백(α-SMA)염색이관찰결혈기육조직내혈관신생정황.결과 (1)종GFP전기인소서적지방조직중가이획득대량GFP-ADSCs,병표체간세포표면분자표기Sca-1[(80.80±0.23)％]화CD44[(77.50 ±0.16)％];(2)SPIO가이유효지표기GFP-ADSCs,재25 mg/L농도범위내불영향GFP-ADSCs적증식;(3)SPIO표기적GFP-ADSCs가이피MRI검측도;(4)3주후SPIO표기적GFP-ADSCs치료조화GFP-ADSCs치료조결혈적기육조직내가견교다적신생혈관,평균혈관밀도분별위4.39±0.76화4.62 ±0.87,신생혈관불부표체내피세포적특이성표기vWF,동시표체GFP,차미혈관밀도수현저고우PBS조(2.15±0.53,P＜0.05),단치료조간차이무통계학의의(P＜0.05).결론 종GFP전기인소서적지방조직중가이획취대량적GFP-ADSCs,기능구촉진소서하지결혈기육조직내적혈관신생,SPIO가이유효적표기GFP-ADSCs,차불영향기증식,기자대적록색형광단백GFP화SPIO가이동시동태시종ADSCs재결혈기육내적존활、천이급귀소정황.
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 × 1010/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 new vessles of treat group express vWF of endotheliocyte specific antigen and GFP, while green fluorescent cannot be found in PBS group.Conclusion A lot of GFP-ADSCs can be isolated from green fluorescent protein transgenic mice, and they could facilitate rebirh vessel in muscle of hind-limb ischemic mice.GFP-ADSCs could be labeled effectively and proliferated with SPIO insusceptibly.The survival, migration and homing of cells with SPIO in receptor could be monitored in vivo by their own fluorescence and MRI.