荧光活性染料DiO标记腹腔巨噬细胞的示踪与应用研究

Study on Tracing and Application of Fluorescent Dye DiO Labelling Peritoneal Macrophages

目的以细胞膜绿色荧光活性染料DiO(DiOC_(18)(3))标记腹腔巨噬细胞(peritoneal macrophage),探讨在巨噬细胞消失反应(macrophage disappearance reaction,MDR)中腹腔巨噬细胞的示踪研究。方法DiO标记腹腔巨噬细胞,过继移植给C57BL/6小鼠;以脂多糖(lipopolysaccharide,LPS)诱导体内MDR。采用荧光显微镜和流式细胞术检测DiO标记的腹腔巨噬细胞数量及荧光强度;分离收集小鼠的各组织,进行冰冻切片,检测DiO标记的腹腔巨噬细胞分布情况。结果荧光显微镜和流式细胞仪观察发现,腹腔注射LPS能显著降低腹腔中DiO标记的腹腔巨噬细胞数量及荧光强度。在MDR过程中消失的腹腔巨噬细胞,通过冰冻切片发现在肝脏、胸腺及脾脏中有分布。结论DiO标记对腹腔巨噬细胞的存活无影响且能长效保持荧光,是一种安全、有效的示踪腹腔巨噬细胞分布的技术手段。

Objective In homeostatic conditions the peritoneal cavity is populated by resident macrophages.Inflammatory stimuli trigger a phenomenon called macrophage disappearance reaction(MDR),during which resident macrophages become irretrievable from the lavage of the serous cavity.This phenomenon was already observed after different inflammatory insults,but is still incompletely understood.MDR can be associated with cell death,adhesion to neighbouring tissues or migration to the draining lymph nodes or the omentum.MDR is a strategy to face and annihilate the infection by which macrophages,under the control of GATA6,move from the peritoneum to the closest tissues in order to alert the immune system.However,the specific distribution of peritoneal macrophages in MDR is still unclear.In our study,peritoneal macrophages labelled with cell membrane green fluorescent dye DiO were used to study the tracking of peritoneal macrophages in the macrophages disappearance reaction.Methods Peritoneal macrophages labelled with DiO were transplanted to C57BL/6mice.The macrophage disappearance reaction was induced by lipopolysaccharide(LPS)in vivo.Fluorescence microscope and flow cytometry were used to detect the number and fluorescence intensity of DiO-labelled peritoneal macrophages.The tissues of mice were separated and collected,and frozen sections were made to detect the distribution of DiO-labelled peritoneal macrophages.Results The observation by fluorescence microscope and flow cytometry showed that intraperitoneal injection of LPS could significantly reduce the number and fluorescence intensity of DiO-labelled peritoneal macrophages.Peritoneal macrophages that disappeared during the macrophage disappearance reaction were found distributed in the liver,thymus and spleen by frozen sections.DiO labelling peritoneal macrophages does not affect cell viability and with long-term stability in vivo,indicating that DiO may be a safe and effective green fluorescent dye for tracking the distribution of peritoneal macrophages.Conclusion This research method will provide a convenient and effective experimental means for exploring the dynamic changes and related biological phenomena of peritoneal macrophages during MDR.Furthermore,it laid a foundation for further research on the causes and mechanisms of MDR.