一种骨髓单个核细胞新型示踪方法——细胞膜染料DiD标记法  

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

作　　者：王芬[1] 谢成颖[2] 曲莹[1] 魏丹[2] 索元震[2] 吴梦瑶[1] 袁燕[1] 魏勋斌[2] 陈彤[1] 

机构地区：[1]复旦大学附属华山医院血液科,上海200040 [2]上海交通大学生物医学工程学院Med-X研究院,上海200030

出　　处：《复旦学报（医学版）》2016年第6期691-696,共6页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81561138002;91542109);上海市基础研究重点项目(13JC1406404);上海市优秀学术带头人计划(16XD1400600)~~

摘　　要：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。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.

关 键 词：细胞膜染料 骨髓单个核细胞 造血干细胞移植 

分 类 号：R457.7[医药卫生—治疗学]