Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

5-羧基荧光素琥珀酰亚胺酯直观检测经交联处理异体组织的交联程度研究

利用荧光技术创建一种新的方法评价经交联处理异体组织的交联程度。以新西兰兔胸主动脉作为研究对象,按与其作用的戊二醛(GA)浓度不同分为空白对照组--未与GA作用(A组),GA浓度0.625%组(B组),GA浓度1.25%组(C组),GA浓度2.5%组(D组),各组与GA交联作用后经5-羧基荧光素琥珀酰亚胺酯(5-FAMSE)处理,然后在荧光显微镜下照相,照片进行荧光强度分析(应用专业图像分析软件--Image-Pro Plus 6.0),结果A组>B组>C组>D组,各组之间差异具有显著统计学意义(P<0.01)。各组同时取一定量血管组织经GA交联后提取其组织蛋白,进行传统的聚丙烯酰胺凝胶电泳(PGE)与5-FAMSE法进行对照,结果组织蛋白提取量A组>B组/C组/D组,无法定量比较B、C、D各组间差异。以上可说明5-FAMSE是一种有效的荧光试剂,可以清楚地反映小血管氨基残端的变化,可以更为方便且直观的评价经GA处理的小血管的交联程度。

Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina

Background Endothelial progenitor cells （EPCs） transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester （CFSE） in laser-injured mouse retina. Methods EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 μmol/L CFSE at 37℃ was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.Results EPCs labeled with 5 μmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks, The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.Conclusion EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.