一种骨髓单个核细胞新型示踪方法——细胞膜染料DiD标记法

 目的 检测细胞膜染料DiD对骨髓单个核细胞(bone marrow mononuclear cells, BM-MNC)的染色效率和造血功能的影响，并用DiD示踪BM-MNC活体直观观察DiD标记的BM-MNC在小鼠颅骨骨髓内的顺利归巢。方法 体外流式细胞术分析方法和激光共聚焦扫描显微镜成像方法检测BM-MNC的DiD染色效率；CCK-8细胞活性-增殖实验和造血集落形成实验，检测DiD染料对BM-MNC的造血功能的影响；单/双光子共聚焦显微镜下活体直观观察DiD标记的BM-MNC在小鼠颅骨骨髓内的归巢。结果 BM-MNC的DiD染色效率高达90%以上，且DiD对BM-MNC的细胞活性-增殖和造血能力均无明显的毒性影响(P＞0.05)；单/双光子共聚焦显微镜下，在活体小鼠颅骨骨髓内可直观观察到DiD标记的BM-MNC的顺利归巢。结论 细胞膜染料DiD对BM-MNC的染色效率高、均一，无明显细胞毒性，荧光不易被淬灭，且受机体自身组织的背景干扰少，用于活体示踪时DiD标记的BM-MNC可顺利归巢到骨髓。

A new tracing method of bone marrow mononuclear cells by cell membrane dye DiD

Objective To detect the labeling efficiency of DiD on bone marrow mononuclear cells (BM-MNC) and the effects of hematopoiesis on BM-MNC imposed by DiD, and then directly observe the successful homing of transplanted BM-MNC labeled by DiD in a live mouse skull marrow. Methods We detected the labeling efficiency of DiD on BM-MNC by in vitro flow cytometry and confocal laser scanning microscopy. We also detected the effects of DiD hematopoiesis on BM-MNC by CCK8 test and colony forming assay, and directly observed the homing of transplanted BM-MNC labeled by DiD in a live mouse skull marrow under the single/two-photon confocal laser scanning microscope. Results The labeling efficiency of DiD on BM-MNC was up to 90%, and the cell vitality-proliferation and capacity to produce blood cells of BM-MNC were changed very little after being labeled by DiD (P＞0.05). The transplanted BM-MNC labeled by DiD could be seen successfully homing to skull marrow in a live mouse under the single/two-photon confocal laser scanning microscope. Conclusions DiD has high and uniform labeling efficiency, non-toxic to cell functions, low rate of fluorescence quenching, low interference from autofluorescence of tissue, and the BM-MNC labeled by DiD could home to bone marrow successfully in a live mouse.