脓毒症早期骨髓血管内皮系统改变的初步研究

Changes of bone marrow vascular system and endothelial cells in the early stage of sepsis

目的拟通过动物实验阐明脓毒症早期骨髓血管内皮系统在组织、细胞层面以及转录水平发生的变化。方法通过盲肠结扎穿刺(cecal ligation and puncture,CLP)建立小鼠脓毒症模型,利用HE染色、组织免疫荧光、流式细胞分析以及实时荧光定量PCR等技术分析骨髓血管结构、通透性、血管内皮细胞相对比例和血管内皮细胞活化基因mRNA表达情况。结果骨髓血管系统在脓毒症早期发生一系列适应性改变。组织学分析表明,骨髓血管结构发生明显重塑,CLP组小鼠骨髓血窦平均密度为(410.43±72.63)个/mm^(2),较假手术组(294.43±68.94)个/mm^(2)明显增加(P<0.01);管腔面积占比为(43.46±3.21)%,显著高于假手术组[(30.28±4.44)%,P<0.001];CLP组小鼠骨髓组织中伊文思蓝含量为(0.42±0.12)ng/mg组织,显著高于假手术组[(0.24±0.09)ng/mg组织,P<0.05],提示血管通透性升高。流式细胞分析结果表明,CLP组小鼠骨髓内皮细胞呈现增殖状态,Ki67^(+)内皮细胞比例升高[(1.91±0.65)%比(5.06±1.10)%,P<0.01];转录水平分析表明,与血管内皮细胞活化有关的部分基因mRNA水平上调。结论早期脓毒症改变了骨髓血管和血管内皮细胞的结构与功能,对骨髓微环境产生了较为显著的影响。

Objective To clarify the changes in the bone marrow vascular system in the early stage of sepsis in animal model.Methods A sepsis mouse model was established by cecal ligation and puncture(CLP),and HE staining,immunofluorescence staining,flow cytometry and real-time quantitative PCR were used to comprehensively analyze the varieties of bone marrow vascular system in structure,the relative proportion of vascular endothelial cells and the expressions of damage-related genes at mRNA level.Results A series of adaptive changes occurred in the bone marrow vascular system in the early stage of sepsis.Histological analysis showed that the bone marrow vascular structure was significantly remodeled.The average density of bone marrow sinusoids in the CLP group was(410.43±72.63)counts/mm^(2),which was significantly higher than that in the sham group[(294.43±68.94)counts/mm^(2),P<0.01].The area of luminal pixels accounted for(43.46±3.21)%,which was significantly higher than that in the sham group[(30.28±4.44)%,P<0.001].The exudation amount of evans blue in the bone marrow tissue of the CLP group was(0.42±0.12)ng/mg tissue,which was significantly higher than that in the sham group[(0.24±0.09)ng/mg tissue,P<0.05],suggesting increased vascular permeability.The results of flow cytometry analysis showed that the EC in bone marrow of the CLP group mice was in a proliferative state,with the proportion of Ki67^(+) endothelial cell increasing[(1.91±0.65)%vs.(5.06±1.10)%,P<0.01].The mRNA levels of some genes related to the activation of vascular endothelial cells were up-regulated.Conclusion Sepsis changes the structure and function of the bone marrow vascular system,and has a significant impact on the bone marrow microenvironment.