羧基荧光素乙酰乙酸在移植免疫研究中的应用

Application of vital dye CFDA-SE in transplant immune research

目的:介绍和探讨活体荧光示踪染料羧基荧光素乙酰乙酸(CFDA SE)在移植免疫研究中的应用及价值。 方法:以不同浓度的CFDA SE标记小鼠脾细胞用于体内外实验。标记Balb/c(H2d)和C57BL/6小鼠(H2b)脾细胞 行混合淋巴细胞培养,流式细胞术观察标记细胞的增殖情况。Balb/c裸小鼠(H2d)构建移植物抗宿主(GVHR)、宿 主抗移植物(HVGR)模型;通过流式细胞术检测标记细胞在外周血及脾的增殖情况。 结果:荧光显微镜观察到 标记细胞。流式细胞术检测到标记细胞荧光强度出现倍减现象,表明细胞发生了增殖;无抗原刺激组荧光强度无 明显变化,抗原刺激后24～72h标记细胞停留在脾,第3天后增殖细胞进入外周血液。 结论:羧基荧光素琥珀酰 亚胺酯(CFSE)标记技术可应用于体内、体外实验,有助于对细胞增殖及定位进行检测,在移植免疫实验研究中具 有很高的应用价值。

Objective: To introduce and investigate the application of Carboxy-fluorescein diacetate succinimidyl (CFDA-SE) in transplant immune research. Methods: Murine splenocytes and T cells were labeled by CFDA-SE with different concentrations (5, 10, 20 μmol/L, respectively) for mixed lymphocytes culture (MLC) and the studies for graft versus host reaction (GVHR) and host versus graft reaction (HVGR). In GVHR model, splenocytes from C57BL/6 (H2 b) mice were labeled with CFDA-SE and transfused into Balb/c (H2 d) nude mice. In HVGR model, splenocytes from C57BL/6 mice were transfused into Balb/c nude mice that were reconstituted with syngeneic T-cells or splenocytes from Balb/c mice labeled with CFDA-SE, and Balb/c nude mice that only received transfusion with syngeneic CFSE-labeling T-cells or splenocytes were taken as control group. The lymphocytes division, proliferation and histological localization were determinated by flow cytometry and fluorescence microscope. Results:After a certain interval, CFSE labeled splenocytes or T cells were significantly proliferated, and there were multi-generation division and fluorescent strength decreased in the histograms. On the contrary, there was no noticeable change in division or fluorescent intensity in the control groups. During 24 hrs to 72 hrs after antigen stimulating, the labeled cells settled in spleen, and then the mass of expanding cells migrated into peripheral circulation after 72 hrs. The different concentrations did not influence the determination by flow cytometry, but by fluorescence microscopy, the fluorescence appeared stronger following the higher concentration. Conclusion: The technique is applicable to both in vivo division of adoptive transferred cells and in vitro cell division, and it can resolve multiple successive generations using flow cytometry or histological localization by fluorescence microscope.