氯甲基苯甲酰氨荧光染料用于羊骨髓基质干细胞标记和心肌内示踪动物实验

摘要

目的 探讨氯甲基苯甲酰氨荧光染料(CM-DiI)标记羊骨髓基质干细胞(bone marrow stromal cells,BMSCs)进行心肌内移植示踪的可行性.方法 羊BMSCs经CM-DiI标记后(D-BMSCs),体外传代培养观察荧光变化;D-BMSCs细胞悬液经心肌内直接注射于心梗区和梗死移行区,观察示踪有效性.结果 CM-DiI可高效标记羊BMSCs.D-BMSCs在荧光显微镜下呈橙红色.体外培养证实D-BMSCs保持骨髓基质干细胞特异细胞形态,并在传2代后仍保持较强荧光.流式细胞术分析表明D-BMSCs与BMSCs增殖能力没有差异;三系分化证实D-BMSCs与BMSCs分化潜能相当.心肌内植入1周,D-BMSCs在荧光显微镜下发红色荧光,清晰显示其在心脏中的位置和细胞形态.结论 CM-DiI可用于羊BMSCs标记和心肌内移植示踪.%Objective To explore the feasibility of labeling ovine bone marrow stromal cells (BMSCs) with CM-DiI for intracardiac tracking. Methods Ovine BMSCs were labelled by CM-DiI, and then cultured and monitored by fluorescence microscopy. Tracking of BMSCs was done after intramyocardial injection of CM-DiI-labeled BMSCs into infarction and peri-infarction area and examined by histology and fluorescence microscopy. Results BMSCs can be labeled by CM-DiI. The labelled BMSCs (D-BMSCs) showed red auto-fluorescence. In vitro study the D-BMSCs didn't show to alter the morphology of mesenchymal stem cells, and maintain strong fluorescence after subpassage culture. Flow cytometry analysis demonstrates that there is no difference in cell proliferation between D-BMSCs and BMSCs. CM-DiI labeling did not influenced multipotent capacity of BMSCs proved by inducing differentiation into adipose, chondrocyte and osteoblast. One week after intramyocardial transplantation, the D-BMSCs maintain strong fluorescence,clearly showing the location and the morphological characteristics of the implanted BMSCs. Conclusion CM-DiI labeling can be used as a stable fluorescent intracardiac tracking system.